U.S. patent application number 11/580223 was filed with the patent office on 2007-08-02 for selective posttranslational modification of phage-displayed polypeptides.
This patent application is currently assigned to The Scripps Research Institute. Invention is credited to Peter Schultz, Feng Tian, Meng-Lin Tsao.
Application Number | 20070178448 11/580223 |
Document ID | / |
Family ID | 37963059 |
Filed Date | 2007-08-02 |
United States Patent
Application |
20070178448 |
Kind Code |
A1 |
Tsao; Meng-Lin ; et
al. |
August 2, 2007 |
Selective posttranslational modification of phage-displayed
polypeptides
Abstract
The invention relates to posttranslational modification of
phage-displayed polypeptides. These displayed polypeptides comprise
at least one unnatural amino acid, e.g., an aryl-azide amino acid
such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as
para-propargyloxyphenylalanine, which are incorporated into the
phage-displayed fusion polypeptide at a selected position by using
an in vivo orthogonal translation system comprising a suitable
orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA
species. These unnatural amino acids advantageously provide targets
for posttranslational modifications such as azide-alkyne [3+2]
cycloaddition reactions and Staudinger modifications.
Inventors: |
Tsao; Meng-Lin; (San Diego,
CA) ; Tian; Feng; (San Diego, CA) ; Schultz;
Peter; (La Jolla, CA) |
Correspondence
Address: |
QUINE INTELLECTUAL PROPERTY LAW GROUP, P.C.
P O BOX 458
ALAMEDA
CA
94501
US
|
Assignee: |
The Scripps Research
Institute
La Jolla
CA
|
Family ID: |
37963059 |
Appl. No.: |
11/580223 |
Filed: |
October 11, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60726137 |
Oct 12, 2005 |
|
|
|
60737622 |
Nov 16, 2005 |
|
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Current U.S.
Class: |
435/5 ;
435/235.1 |
Current CPC
Class: |
C12N 2795/14122
20130101; C12P 21/02 20130101; C12N 7/00 20130101; C07K 2319/50
20130101; C12N 15/1037 20130101; C12N 9/93 20130101; C07K 14/005
20130101; C12N 15/67 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
435/005 ;
435/235.1 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C12N 7/01 20060101 C12N007/01 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This invention was made with government support from the
Department of Energy under Grant No. ER46051, and the National
Institutes of Health under Grant No. GM56528. The government may
have certain rights to this invention.
Claims
1. A phage comprising a polypeptide, said polypeptide comprising at
least one post-translationally modified unnatural amino acid
residue selected from an aryl-azide unnatural amino acid residue
and an alkynyl unnatural amino acid residue.
2. The phage of claim 1, wherein said phage is a filamentous
phage.
3. The phage of claim 1, wherein said phage is an M13-derived
phage.
4. The phage of claim 1, wherein said polypeptide is a fusion
polypeptide.
5. The phage of claim 4, wherein said fusion polypeptide comprises
a peptide linker protease recognition sequence specifically
cleavable by a site-specific protease.
6. The phage of claim 5, wherein said site-specific protease is
selected from Factor Xa, Factor XIa, Kallikvein, thrombin, Factor
XIIa, collagenase and enterokinase.
7. The phage of claim 1, wherein said aryl-azide unnatural amino
acid residue is a para-azido-L-phenylalanine residue.
8. The phage of claim 1, wherein said alkynyl unnatural amino acid
residue is a para-propargyloxyphenylalanine residue.
9. The phage of claim 1, wherein said post-translationally modified
unnatural amino acid residue is produced by an azide-alkyne [3+2]
cycloaddition reaction.
10. The phage of claim 1, wherein said post-translationally
modified unnatural amino acid residue comprises a triazole
linkage.
11. The phage of claim 1, wherein said post-translationally
modified aryl-azide unnatural amino acid residue is produced by a
Staudinger ligation reaction.
12. The phage of claim 11, wherein said phage is viable.
13. The phage of claim 1, wherein said phage are immobilized to a
solid support.
14. A plurality of the phage of claim 1.
15. A plurality of the phage of claim 1, wherein said plurality of
phage display a plurality of different polypeptides.
16. The plurality of phage of claim 15, wherein said plurality of
phage comprises a phage display library.
17. The phage of claim 1, wherein said phage is purified or
isolated.
18. A method for producing a post-translationally modified phage,
the method comprising: (a) providing a phage comprising a
polypeptide, said polypeptide comprising at least one unnatural
amino acid residue selected from an aryl-azide unnatural amino acid
residue and an alkynyl unnatural amino acid residue; and (b)
reacting said phage under conditions wherein said unnatural amino
acid residue undergoes covalent modification, thereby producing a
post-translationally modified phage.
19. The method of claim 18, wherein said reacting step comprises an
azide-alkyne [3+2] cycloaddition reaction to produce a
post-translationally modified phage.
20. The method of claim 18, wherein said unnatural amino acid
residue is an aryl-azide unnatural amino acid residue, and said
reacting step comprises a Staudinger ligation reaction to produce a
post-translationally modified phage.
21. The method of claim 20, wherein said post-translationally
modified phage produced by said method is viable.
22. The method of claim 18, wherein said providing step comprises
providing a phage comprising at least one aryl-azide unnatural
amino acid residue, wherein said aryl-azide unnatural amino acid
residue is a para-azido-L-phenylalanine residue.
23. The method of claim 18, wherein said providing step comprises
providing a phage comprising at least one alkynyl unnatural amino
acid residue, wherein said alkynyl unnatural amino acid residue is
a para-propargyloxyphenylalanine residue.
24. The method of claim 18, wherein said providing step comprises
(i) providing a eubacterial host cell comprising: A) a nucleic acid
molecule encoding said phage, said nucleic acid molecule comprising
a polynucleotide subsequence encoding said polypeptide, said
polynucleotide subsequence comprising at least one selector codon;
B) a nucleic acid molecule encoding an aminoacyl-tRNA synthetase
that is orthogonal in said host cell (O-RS); C) a nucleic acid
molecule encoding a tRNA that is orthogonal in said host cell
(O-tRNA), wherein said O-RS preferentially aminoacylates said
O-tRNA with said unnatural amino acid in said host cell and wherein
said selector codon is recognized by said O-tRNA; and D) an
aryl-azide or alkynyl unnatural amino acid; and (ii) culturing said
host cell, thereby producing a polypeptide encoded by said
polynucleotide subsequence, where said aryl-azide or alkynyl
unnatural amino acid is incorporated into said polypeptide during
translation in response to the selector codon, and producing a
phage comprising a polypeptide encoded by said polynucleotide
subsequence, where said aryl-azide or alkynyl unnatural amino acid
is incorporated into said polypeptide.
25. The method of claim 24, wherein said providing step comprises
providing an E. coli host cell.
26. The method of claim 24, wherein said providing step comprises
providing a eubacterial host cell comprising a nucleic acid
molecule encoding an O-RS, wherein said O-RS is derived from a
Methanococcus jannaschii aminoacyl-tRNA synthetase.
27. The method of claim 24, wherein said providing step comprises
providing a eubacterial host cell comprising a nucleic acid
molecule encoding an O-RS, wherein O-RS is derived from a
Methanococcus jannaschii tyrosyl-tRNA synthetase.
28. The method of claim 24, wherein said providing step comprises
providing a eubacterial host cell comprising a nucleic acid
molecule encoding an O-tRNA, wherein said O-tRNA is an amber
suppressor tRNA.
29. A phage comprising a polypeptide, said polypeptide comprising
at least one post-translationally modified unnatural amino acid
residue.
30. The phage of claim 29, wherein said phage is viable.
31. The phage of claim 29, wherein said unnatural amino acid
residue is selected from para-propargyloxyphenylalanine,
para-azido-L-phenylalanine, para-acetyl-L-phenylalanine,
meta-acetyl-L-phenylalanine, para-(3-oxobutanoyl)-L-phenylalanine,
para-(2-amino-1-hydroxyethyl)-L-phenylalanine,
para-isopropylthiocarbonyl-L-phenylalanine and
para-ethylthiocarbonyl-L-phenylalanine.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to and benefit of U.S.
Provisional Patent Application Ser. No. 60/726,137, filed on Oct.
12, 2005, and Provisional Patent Application Ser. No. 60/737,622,
filed on Nov. 16, 2005, the contents of which are hereby
incorporated by reference in their entirety for all purposes.
FIELD OF THE INVENTION
[0003] The invention relates to the field of protein chemistry,
e.g., translation biochemistry. The invention relates to
compositions and methods for making bacteriophage, where the phage
comprise a displayed polypeptide having an unnatural amino acid
that can serve as a target for selective covalent posttranslational
modification, resulting in a posttranslationally modified
phage.
BACKGROUND OF THE INVENTION
[0004] The study of protein structure and function has historically
relied upon the reaction chemistries that are available using the
reactive groups of the naturally occurring amino acids.
Unfortunately, every known organism, from bacteria to humans,
encodes the same twenty common amino acids (with the rare
exceptions of selenocysteine (see, e.g., A. Bock et al., (1991),
Molecular Microbiology 5:515-20) and pyrrolysine (see, e.g., G.
Srinivasan, et al., (2002), Science 296:1459-62). This limited
selection of R-groups has restricted the study of protein structure
and function, where the studies are confined by the chemical
properties of the naturally occurring amino acids.
[0005] The limiting number of natural amino acids restricts the
ability to make highly targeted posttranslational protein
modifications to the exclusion of all other amino acids in a
protein. Most modification reactions currently used in the art
involve covalent bond formation between nucleophilic and
electrophilic reaction partners that target the naturally occurring
nucleophilic residues in the protein amino acid side chains, e.g.,
the reaction of .alpha.-halo ketones with histidine or cysteine
side chains. Selectivity in these cases is determined by the number
and accessibility of the nucleophilic residues in the protein.
Unfortunately, naturally occurring proteins frequently contain
poorly positioned (e.g., inaccessible) reaction sites or multiple
reaction targets (e.g., lysine, histidine and cysteine residues),
resulting in poor selectivity in the modification reactions, making
highly targeted protein modification by nucleophilic/electrophilic
reagents difficult. Furthermore, the sites of modification are
typically limited to the naturally occurring nucleophilic side
chains of lysine, histidine or cysteine. Modification at other
sites is difficult or impossible.
[0006] Alternative approaches for selectively modifying proteins
with synthetic agents and probes, and covalent attachment of
proteins to surfaces have been attempted. These include
semisynthesis (Muir, Annu. Rev. Biochem. 2003, 72, 249-289), the
use of electrophilic reagents that selectively label cysteine and
lysine residues (Chilkoti et al., Bioconjugate Chem. 1994, 5,
504-507; Rosendahl et al., Bioconjugate Chem. 2005, 16, 200-207),
and the selective introduction of amino acids with reactive side
chains into proteins by in vitro biosynthesis with chemically
aminoacylated tRNAs (Bain et al., J. Am. Chem. Soc. 1989, 111,
8013-8014; Ellman et al., Methods Enzymol. 1991, 202, 301-336).
Each of these approaches suffers from either a lack of target
specificity or other impracticalities.
[0007] One strategy to overcome the limitations of the existing
genetic repertoire is to add amino acids that have distinguishing
chemical properties to the genetic code. This approach has proven
feasible using orthogonal tRNA molecules and corresponding novel
orthogonal aminoacyl-tRNA synthetases to add unnatural amino acids
to proteins using the in vivo protein biosynthetic machinery of a
host cell, e.g., the eubacteria Escherichia coli (E. coli). This
approach is described in various sources, for example, Chin et al.,
Science (2003) 301:964-967; Zhang et al., Proc. Natl. Acad. Sci.
U.S.A. 2004, 101:8882-8887; Anderson et al., Proc. Natl. Acad. Sci.
U.S.A. 2004, 101:7566-7571; Wang et al., (2001) Science
292:498-500; Chin et al., (2002) Journal of the American Chemical
Society 124:9026-9027; Chin and Schultz, (2002) ChemBioChem
11:1135-1137; Chin, et al., (2002) PNAS United States of America
99:11020-11024; Wang and Schultz, (2002) Chem. Comm., 1-10; Wang
and Schultz "Expanding the Genetic Code," Angewandte Chemie Int.
Ed., 44(1):34-66 (2005); and Xie and Schultz, "An Expanding Genetic
Code," Methods 36:227-238 (2005). See also, International
Publications WO 2002/086075, entitled "METHODS AND COMPOSITIONS FOR
THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS;"
WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO
ACIDS;" WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC
CODE;" WO 2005/019415, filed Jul. 7, 2004; WO2005/007870, filed
Jul. 7, 2004; WO 2005/007624, filed Jul. 7, 2004; and International
Publication No. WO2006/034332, filed on Sep. 20, 2005.
[0008] Phage display technology is a malleable and widely utilized
technique that has found applications in diverse biological
disciplines. See, e.g., Smith and Petrenko, Chem. Rev., 97:391-410
(1997); Sidhu, Bimolecular Engineering 18:57-63 (2001); Rodi and
Makowski, Current Opinion in Biotechnology 10:87-93 (1999); and
Willats, Plant Molecular Biology 50:837-854 (2002). For example,
phage display has proven very useful for the isolation of
high-affinity ligands and receptors from large polypeptide
libraries. It has the advantages that large libraries can be easily
generated by recombinant methods, library members can be amplified
for iterative rounds of enrichment, and primary structure can be
determined by DNA sequencing. However, like proteins in general,
phage-displayed peptide libraries are also restricted to the common
20 amino acid building blocks, limiting the functional groups that
can be targeted for posttranslational modification. Moreover,
methods for posttranslational modification of phage-displayed
polypeptides, where the modification reaction uses
physiologically-compatible conditions that preserve protein
activity and phage viability present even greater challenge (Leieux
and Bertozzi (1998) TIBTECH, 16:506).
[0009] In an attempt to expand the scope of phage-display utility,
Noren and co-workers incorporated selenocysteine into phage
displayed peptides using a natural selenocysteine opal suppressing
tRNA (Sandman et al., J. Am. Chem. Soc. (2000) 122:960-961).
Roberts et al. attempted to generalize this approach to peptide
libraries containing other unnatural amino acids using in vitro
mRNA display (Li et al., J. Am. Chem. Soc., (2002) 124:9972) with
chemically aminoacylated amber suppressor tRNAs (Noren et al.,
Science (1989) 244:182-188). However, the generation of a large
number of such tRNAs is impractical, and they are consumed
stoichiometrically.
[0010] What is needed in the art are new strategies for
incorporation of unnatural amino acids into phage-displayed
polypeptides for the purpose of modifying and studying protein
structure and function, where the unnatural amino acids in the
displayed polypeptides can be selectively targeted for
posttranslational modification while displayed on the phage. There
is a need in the art for the creation of new strategies for protein
modification reactions that modify phage-displayed proteins in a
highly selective fashion, and furthermore, allow the modification
of the phage-displayed proteins under physiological conditions that
preserve phage viability following the modification reaction. What
is needed in the art are novel methods for producing targeted
protein modifications on phage-displayed proteins, where the
modifications are highly specific, e.g., modifications where none
of the naturally occurring amino acids in the polypeptides are
subject to cross reactions or side reactions. The invention
described herein fulfills these and other needs, as will be
apparent upon review of the following disclosure.
SUMMARY OF THE INVENTION
[0011] There is a need for chemical reactions that modify proteins,
e.g., phage-displayed proteins, in a highly selective fashion. Most
reactions currently used in the art for the selective modification
of proteins have poor selectivity and are limited to naturally
occurring amino acid residues. The present invention provides
solutions to these problems.
[0012] The invention provides systems for the programmed,
site-specific biosynthetic incorporation of unnatural amino acids
into phage-displayed proteins by manipulating orthogonal
translation systems to work in conjunction with recombinant phage
expression reagents. The invention provides methods for the
subsequent targeted modification of those unnatural amino acid
residues that are incorporated into phage-displayed polypeptides.
The invention provides novel compositions (e.g., phage comprising
various posttranslational modifications) and novel methods for the
generation of posttranslationally modified phage.
[0013] The phage-production systems provided herein take advantage
of orthogonal translation systems that use E. coli host cells for
the selective incorporation of unnatural amino acids into
phage-displayed polypeptides, and the subsequent modification of
those polypeptides using selective modification of the unnatural
amino acid residue. Various chemistries for the modification of the
unnatural amino acid residue in the phage-displayed polypeptide are
demonstrated, including [3+2] cycloaddition reactions and
Staudinger ligations. The nature of the material that is conjugated
to the phage-displayed protein via an unnatural amino acid target
is not particularly limited and can be any desired entity.
[0014] The invention provides phage having a displayed fusion
polypeptide, where the polypeptide comprises at least one
post-translationally modified unnatural amino acid residue. A
variety of reactive unnatural amino acids can be used in the
displayed polypeptide. For example, the unnatural amino acid can be
an aryl-azide unnatural amino acid (e.g.,
para-azido-L-phenylalanine) or an alkynyl unnatural amino acid
(e.g., para-propargyloxyphenylalanine). The phage can be a
filamentous phage, e.g., an M13-derived phage.
[0015] The displayed polypeptide is generally a fusion polypeptide
that comprises a phage capsid protein (or a portion or variant
thereof) and an amino acid sequence of interest. In some
embodiments, the fusion polypeptide is designed to incorporate a
peptide linker protease recognition sequence specifically cleavable
by a site-specific protease, e.g., Factor Xa, Factor XIa,
Kallikvein, thrombin, Factor XIIa, collagenase or enterokinase.
[0016] Various types of modification reactions are employed for the
modification of the phage-displayed polypeptide having the
unnatural amino acid. For example, an azide-alkyne [3+2]
cycloaddition reaction (which produces a triazole linkage) or a
Staudinger ligation reaction can be used. Because of the unique
reaction chemistries of aryl-azide and alkynyl unnatural amino
acids, phage-displayed proteins into which they are incorporated
can be modified with extremely high selectivity. In some cases, the
unnatural amino acid reactive group has the advantage of being
completely alien to in vivo systems, thereby improving reaction
selectivity. Advantageously, use of the Staudinger reaction
preserves viral infectivity.
[0017] The modified phage of the invention can optionally be
immobilized to a solid support. In some embodiments, the phage
comprise a phage polypeptide library, where a plurality of
polypeptides are expressed by the phage. This plurality of phage is
also a feature of the invention. The phage of the invention can be
purified or isolated
[0018] In other embodiments, the invention provides methods for the
production of the aforementioned post-translationally modified
phages. Generally, these methods have the steps of (a) providing a
phage comprising a displayed polypeptide comprising at least one
unnatural amino acid residue that is an aryl-azide unnatural amino
acid residue (e.g., para-azido-L-phenylalanine) or an alkynyl
unnatural amino acid residue (e.g.,
para-propargyloxyphenylalanine); and (b) reacting the phage under
conditions wherein the unnatural amino acid residue undergoes
covalent modification, thereby producing a post-translationally
modified phage. These modification reactions can use an
azide-alkyne [3+2] cycloaddition reaction or a Staudinger ligation
reaction. When the Staudinger modification reaction is used, the
resulting modified phage can be viable virion.
[0019] More specifically, providing the unmodified phage can have
the following steps: (i) providing a eubacterial host cell that
comprises (A) a nucleic acid molecule encoding the phage, where the
polynucleotide portion that encodes the fusion polypeptide of
interest comprises at least one selector codon; (B) a nucleic acid
molecule encoding an aminoacyl-tRNA synthetase that is orthogonal
in said host cell (O-RS); (C) a nucleic acid molecule encoding a
tRNA that is orthogonal in the host cell (O-tRNA), wherein the O-RS
preferentially aminoacylates the O-tRNA with the unnatural amino
acid in the host cell and where said selector codon is recognized
by the O-tRNA; and D) an aryl-azide or an alkynyl unnatural amino
acid; and (ii) culturing the host cell, thereby producing a
polypeptide encoded by said polynucleotide subsequence, where an
aryl-azide or an alkynyl unnatural amino acid is incorporated into
the polypeptide during translation in response to the selector
codon, and producing a phage comprising a polypeptide encoded by
said polynucleotide subsequence, where an aryl-azide or an alkynyl
unnatural amino acid is incorporated into said polypeptide. In some
aspects, an E. coli host cell is also provided.
[0020] The orthogonal tRNA and synthetase that are used in the
methods is not particularly limiting. In some embodiments, the host
cell comprises a nucleic acid molecule that encodes an O-RS derived
from a Methanococcus jannaschii aminoacyl-tRNA synthetase, e.g., a
Methanococcus jannaschii tyrosyl-tRNA synthetase. In some
embodiments, the O-tRNA used is an amber suppressor tRNA.
[0021] In still other embodiments, it is further contemplated that
additional unnatural amino acids can be used to target phage for
post-translational modifications, where the unnatural amino acid is
incorporated into the phage by using an orthogonal translation
system comprising a suppressor tRNA and mutant synthetase.
[0022] In some aspects, any phage comprising a polypeptide that
comprises at least one post-translationally modified unnatural
amino acid residue is a phage of the invention, where the at least
one unnatural amino acid allows for targeted covalent modification.
In some aspects, the phage are viable following post-translational
modification. Reactive amino acids that can be incorporated into
phage in this manner can include para-propargyloxyphenylalanine,
para-azido-L-phenylalanine, para-acetyl-L-phenylalanine,
meta-acetyl-L-phenylalanine, para-(3-oxobutanoyl)-L-phenylalanine,
para-(2-amino-1-hydroxyethyl)-L-phenylalanine,
para-isopropylthiocarbonyl-L-phenylalanine and
para-ethylthiocarbonyl-L-phenylalanine.
DEFINITIONS
[0023] Before describing the invention in detail, it is to be
understood that this invention is not limited to particular
biological systems, which can, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting. As used in this specification and the appended claims,
the singular forms "a", "an" and "the" include plural referents
unless the content clearly dictates otherwise. Thus, for example,
reference to "a cell" includes combinations of two or more cells;
reference to "a polynucleotide" includes, as a practical matter,
many copies of that polynucleotide.
[0024] Unless defined herein and below in the reminder of the
specification, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in
the art to which the invention pertains.
[0025] Orthogonal: As used herein, the term "orthogonal" refers to
a molecule (e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal
aminoacyl-tRNA synthetase (O-RS)) that functions with endogenous
components of a cell with reduced efficiency as compared to a
corresponding molecule that is endogenous to the cell or
translation system, or that fails to function with endogenous
components of the cell. In the context of tRNAs and aminoacyl-tRNA
synthetases, orthogonal refers to an inability or reduced
efficiency, e.g., less than 20% efficiency, less than 10%
efficiency, less than 5% efficiency, or less than 1% efficiency, of
an orthogonal tRNA to function with an endogenous tRNA synthetase
compared to an endogenous tRNA to function with the endogenous tRNA
synthetase, or of an orthogonal aminoacyl-tRNA synthetase to
function with an endogenous tRNA compared to an endogenous tRNA
synthetase to function with the endogenous tRNA. The orthogonal
molecule lacks a functionally normal endogenous complementary
molecule in the cell. For example, an orthogonal tRNA in a cell is
aminoacylated by any endogenous RS of the cell with reduced or even
zero efficiency, when compared to aminoacylation of an endogenous
tRNA by the endogenous RS. In another example, an orthogonal RS
aminoacylates any endogenous tRNA a cell of interest with reduced
or even zero efficiency, as compared to aminoacylation of the
endogenous tRNA by an endogenous RS. A second orthogonal molecule
can be introduced into the cell that functions with the first
orthogonal molecule. For example, an orthogonal tRNA/RS pair
includes introduced complementary components that function together
in the cell with an efficiency (e.g., 45% efficiency, 50%
efficiency, 60% efficiency, 70% efficiency, 75% efficiency, 80%
efficiency, 90% efficiency, 95% efficiency, or 99% or more
efficiency) as compared to that of a control, e.g., a corresponding
tRNA/RS endogenous pair, or an active orthogonal pair (e.g., a
tyrosyl orthogonal tRNA/RS pair).
[0026] Orthogonal tyrosyl-tRNA: As used herein, an orthogonal
tyrosyl-tRNA (tyrosyl-O-tRNA) is a tRNA that is orthogonal to a
translation system of interest, where the tRNA is: (1) identical or
substantially similar to a naturally occurring tyrosyl-tRNA, (2)
derived from a naturally occurring tyrosyl-tRNA by natural or
artificial mutagenesis, (3) derived by any process that takes a
sequence of a wild-type or mutant tyrosyl-tRNA sequence of (1) or
(2) into account, (4) homologous to a wild-type or mutant
tyrosyl-tRNA; (5) homologous to any example tRNA that is designated
as a substrate for a tyrosyl-tRNA synthetase in FIG. 1 or 2, or (6)
a conservative variant of any example tRNA that is designated as a
substrate for a tyrosyl-tRNA synthetase in FIG. 1 or 2. The
tyrosyl-tRNA can exist charged with an amino acid, or in an
uncharged state. It is also to be understood that a
"tyrosyl-O-tRNA" optionally is charged (aminoacylated) by a cognate
synthetase with an amino acid other than tyrosine or leucine,
respectively, e.g., with an unnatural amino acid. Indeed, it will
be appreciated that a tyrosyl-O-tRNA of the invention is
advantageously used to insert essentially any amino acid, whether
natural or artificial, into a growing polypeptide, during
translation, in response to a selector codon.
[0027] Orthogonal tyrosyl amino acid synthetase: As used herein, an
orthogonal tyrosyl amino acid synthetase (tyrosyl-O-RS) is an
enzyme that preferentially aminoacylates the tyrosyl-O-tRNA with an
amino acid in a translation system of interest. The amino acid that
the tyrosyl-O-RS loads onto the tyrosyl-O-tRNA can be any amino
acid, whether natural, unnatural or artificial, and is not limited
herein. The synthetase is optionally the same as or homologous to a
naturally occurring tyrosyl amino acid synthetase, or the same as
or homologous to a synthetase designated as an O-RS in FIG. 1 or 2
(see, SEQ ID NOS: 4-10). For example, the O-RS can be a
conservative variant of a tyrosyl-O-RS of FIG. 1, and/or can be at
least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in
sequence to an O-RS of FIG. 1.
[0028] Cognate: The term "cognate" refers to components that
function together, e.g., an orthogonal tRNA and an orthogonal
aminoacyl-tRNA synthetase. The components can also be referred to
as being complementary.
[0029] Preferentially aminoacylates: As used herein in reference to
orthogonal translation systems, an O-RS "preferentially
aminoacylates" a cognate O-tRNA when the O-RS charges the O-tRNA
with an amino acid more efficiently than it charges any endogenous
tRNA in an expression system. That is, when the O-tRNA and any
given endogenous tRNA are present in a translation system in
approximately equal molar ratios, the O-RS will charge the O-tRNA
more frequently than it will charge the endogenous tRNA.
Preferably, the relative ratio of O-tRNA charged by the O-RS to
endogenous tRNA charged by the O-RS is high, preferably resulting
in the O-RS charging the O-tRNA exclusively, or nearly exclusively,
when the O-tRNA and endogenous tRNA are present in equal molar
concentrations in the translation system. The relative ratio
between O-tRNA and endogenous tRNA that is charged by the O-RS,
when the O-tRNA and O-RS are present at equal molar concentrations,
is greater than 1:1 , preferably at least about 2:1, more
preferably 5:1, still more preferably 10:1, yet more preferably
20:1, still more preferably 50:1, yet more preferably 75:1, still
more preferably 95:1, 98:1, 99:1, 100:1, 500:1, 1,000:1, 5,000:1 or
higher.
[0030] The O-RS "preferentially aminoacylates an O-tRNA with an
unnatural amino acid" when (a) the O-RS preferentially
aminoacylates the O-tRNA compared to an endogenous tRNA, and (b)
where that aminoacylation is specific for the unnatural amino acid,
as compared to aminoacylation of the O-tRNA by the O-RS with any
natural amino acid. That is, when the unnatural and natural amino
acids are present in equal molar amounts in a translation system
comprising the O-RS and O-tRNA, the O-RS will load the O-tRNA with
the unnatural amino acid more frequently than with the natural
amino acid. Preferably, the relative ratio of O-tRNA charged with
the unnatural amino acid to O-tRNA charged with the natural amino
acid is high. More preferably, O-RS charges the O-tRNA exclusively,
or nearly exclusively, with the unnatural amino acid. The relative
ratio between charging of the O-tRNA with the unnatural amino acid
and charging of the O-tRNA with the natural amino acid, when both
the natural and unnatural amino acids are present in the
translation system in equal molar concentrations, is greater than
1:1, preferably at least about 2:1, more preferably 5:1, still more
preferably 10:1, yet more preferably 20:1, still more preferably
50:1, yet more preferably 75:1, still more preferably 95:1, 98:1,
99:1, 100:1, 500:1, 1,000:1, 5,000:1 or higher.
[0031] Selector codon: The term "selector codon" refers to codons
recognized by the O-tRNA in the translation process and not
recognized by an endogenous tRNA. The O-tRNA anticodon loop
recognizes the selector codon on the mRNA and incorporates its
amino acid, e.g., an unnatural amino acid, at this site in the
polypeptide. Selector codons can include, e.g., nonsense codons,
such as, stop codons, e.g., amber, ochre, and opal codons; four or
more base codons; rare codons; codons derived from natural or
unnatural base pairs and/or the like.
[0032] Suppressor tRNA: A suppressor tRNA is a tRNA that alters the
reading of a messenger RNA (mRNA) in a given translation system,
e.g., by providing a mechanism for incorporating an amino acid into
a polypeptide chain in response to a selector codon. For example, a
suppressor tRNA can read through, e.g., a stop codon (e.g., an
amber, ocher or opal codon), a four base codon, a rare codon,
etc.
[0033] Suppression activity: As used herein, the term "suppression
activity" refers, in general, to the ability of a tRNA (e.g., a
suppressor tRNA) to allow translational read-through of a codon
(e.g., a selector codon that is an amber codon or a 4-or-more base
codon) that would otherwise result in the termination of
translation or mistranslation (e.g., frame-shifting). Suppression
activity of a suppressor tRNA can be expressed as a percentage of
translational read-through activity observed compared to a second
suppressor tRNA, or as compared to a control system, e.g., a
control system lacking an O-RS.
[0034] The present invention provides various methods by which
suppression activity can be quantitated. Percent suppression of a
particular O-tRNA and O-RS against a selector codon (e.g., an amber
codon) of interest refers to the percentage of activity of a given
expressed test marker (e.g., LacZ), that includes a selector codon,
in a nucleic acid encoding the expressed test marker, in a
translation system of interest, where the translation system of
interest includes an O-RS and an O-tRNA, as compared to a positive
control construct, where the positive control lacks the O-tRNA, the
O-RS and the selector codon. Thus, for example, if an active
positive control marker construct that lacks a selector codon has
an observed activity of X in a given translation system, in units
relevant to the marker assay at issue, then percent suppression of
a test construct comprising the selector codon is the percentage of
X that the test marker construct displays under essentially the
same environmental conditions as the positive control marker was
expressed under, except that the test marker construct is expressed
in a translation system that also includes the O-tRNA and the O-RS.
Typically, the translation system expressing the test marker also
includes an amino acid that is recognized by the O-RS and O-tRNA.
Optionally, the percent suppression measurement can be refined by
comparison of the test marker to a "background" or "negative"
control marker construct, which includes the same selector codon as
the test marker, but in a system that does not include the O-tRNA,
O-RS and/or relevant amino acid recognized by the O-tRNA and/or
O-RS. This negative control is useful in normalizing percent
suppression measurements to account for background signal effects
from the marker in the translation system of interest.
[0035] Suppression efficiency can be determined by any of a number
of assays known in the art. For example, a .beta.-galactosidase
reporter assay can be used, e.g., a derivatived lacZ plasmid (where
the construct has a selector codon n the lacZ nucleic acid
sequence) is introduced into cells from an appropriate organism
(e.g., an organism where the orthogonal components can be used)
along with plasmid comprising an O-tRNA of the invention. A cognate
synthetase can also be introduced (either as a polypeptide or a
polynucleotide that encodes the cognate synthetase when expressed).
The cells are grown in media to a desired density, e.g., to an
OD.sub.600 of about 0.5, and .beta.-galactosidase assays are
performed, e.g., using the BetaFluor.TM. .beta.-Galactosidase Assay
Kit (Novagen). Percent suppression can be calculated as the
percentage of activity for a sample relative to a comparable
control, e.g., the value observed from the derivatized lacZ
construct, where the construct has a corresponding sense codon at
desired position rather than a selector codon.
[0036] Translation system: The term "translation system" refers to
the components that incorporate an amino acid into a growing
polypeptide chain (protein). Components of a translation system can
include, e.g., ribosomes, tRNAs, synthetases, mRNA and the like.
The O-tRNA and/or the O-RSs of the invention can be added to or be
part of an in vitro or in vivo translation system, e.g., in a
non-eukaryotic cell, e.g., a bacterium (such as E. coli), or in a
eukaryotic cell, e.g., a yeast cell, a mammalian cell, a plant
cell, an algae cell, a fungus cell, an insect cell, and/or the
like.
[0037] Unnatural amino acid: As used herein, the term "unnatural
amino acid" refers to any amino acid, modified amino acid, -and/or
amino acid analogue, that is not one of the 20 common naturally
occurring amino acids or seleno cysteine or pyrrolysine. For
example, the unnatural amino acid p-azido-L-phenylalanine finds use
with the invention.
[0038] Derived from: As used herein, the term "derived from" refers
to a component that is isolated from or made using a specified
molecule or organism, or information from the specified molecule or
organism. For example, a polypeptide that is derived from a second
polypeptide caninclude an amino acid sequence that is identical or
substantially similar to the amino acid sequence of the second
polypeptide. In the case of polypeptides, the derived species can
be obtained by, for example, naturally occurring mutagenesis,
artificial directed mutagenesis or artificial random mutagenesis.
The mutagenesis used to derive polypeptides can be intentionally
directed or intentionally random, or a mixture of each. The
mutagenesis of a polypepitde to create a different polypeptide
derived from the first can be a random event (e.g., caused by
polymerase infidelity) and the identification of the derived
polypeptide can be made by appropriate screening methods, e.g., as
discussed herein. Mutagenesis of a polypeptide typically entails
manipulation of the polynucleotide that encodes the
polypeptide.
[0039] Positive selection or screening marker: As used herein, the
term "positive selection or screening marker" refers to a marker
that, when present, e.g., expressed, activated or the like, results
in identification of a cell, which comprises the trait, e.g., a
cell with the positive selection marker, from those without the
trait.
[0040] Negative selection or screening marker: As used herein, the
term "negative selection or screening marker" refers to a marker
that, when present, e.g., expressed, activated, or the like, allows
identification of a cell that does not comprise a selected property
or trait (e.g., as compared to a cell that does possess the
property or trait).
[0041] Reporter: As used herein, the term "reporter" refers to a
component that can be used to identify and/or select target
components of a system of interest. For example, a reporter can
include a protein, e.g., an enzyme, that confers antibiotic
resistance or sensitivity (e.g., .beta.-lactamase, chloramphenicol
acetyltransferase (CAT), and the like), a fluorescent screening
marker (e.g., green fluorescent protein (e.g., (GFP), YFP, EGFP,
RFP, etc.), a luminescent marker (e.g., a firefly luciferase
protein), an affinity based screening marker, or positive or
negative selectable marker genes such as lacZ, .beta.-gal/lacZ
(.beta.-galactosidase), ADH (alcohol dehydrogenase), his3, ura3,
leu2, lys2, or the like.
[0042] Eukaryote: As used herein, the term "eukaryote" refers to
organisms belonging to the Kingdom Eucarya. Eukaryotes are
generally distinguishable from prokaryotes by their typically
multicellular organization (but not exclusively multicellular, for
example, yeast), the presence of a membrane-bound nucleus and other
membrane-bound organelles, linear genetic material (i.e., linear
chromosomes), the absence of operons, the presence of introns,
message capping and poly-A mRNA, and other biochemical
characteristics, such as a distinguishing ribosomal structure.
Eukaryotic organisms include, for example, animals (e.g., mammals,
insects, reptiles, birds, etc.), ciliates, plants (e.g., monocots,
dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia,
protists, etc.
[0043] Prokaryote: As used herein, the term "prokaryote" refers to
organisms belonging to the Kingdom Monera (also termed Procarya).
Prokaryotic organisms are generally distinguishable from eukaryotes
by their unicellular organization, asexual reproduction by budding
or fission, the lack of a membrane-bound nucleus or other
membrane-bound organelles, a circular chromosome, the presence of
operons, the absence of introns, message capping and poly-A mRNA,
and other biochemical characteristics, such as a distinguishing
ribosomal structure. The Prokarya include subkingdoms Eubacteria
and Archaea (sometimes termed "Archaebacteria"). Cyanobacteria (the
blue green algae) and mycoplasma are sometimes given separate
classifications under the Kingdom Monera.
[0044] Bacteria: As used herein, the terms "bacteria" and
"eubacteria" refer to prokaryotic organisms that are
distinguishable from Archaea. Similarly, Archaea refers to
prokaryotes that are distinguishable from eubacteria. Eubacteria
and Archaea can be distinguished by a number morphological and
biochemical criteria. For example, differences in ribosomal RNA
sequences, RNA polymerase structure, the presence or absence of
introns, antibiotic sensitivity, the presence or absence of cell
wall peptidoglycans adn other cell wall components, the branched
versus unbranched structures of membrane lipids, and the
presence/absence of histones and histone-like proteins are used to
assign an organism to Eubacteria or Archaea.
[0045] Examples of Eubacteria include Escherichia coli, Thermus
therinophilus and Bacillus stearothermophilus. Example of Archaea
include Methanococcus jannaschii (Mj), Methanosarcina mazei (Mm),
Methanobacterium thermoautotrophicum (Mt), Methanococcus
maripaludis, Methanopyrus kandleri, Halobacterium such as Haloferax
volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus
(Af), Pyrococcus furiosus (Pf), Pyrococcus horikoshii (Ph),
Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus
(Ss), Sulfolobus tokodaii, Aeuropyrum pernix (Ap), Thermoplasma
acidophilum and Thermoplasma volcanium.
[0046] Conservative variant: As used herein, the term "conservative
variant," in the context of a translation component, refers to a
translation component, e.g., a conservative variant O-tRNA or a
conservative variant O-RS, that functionally performs similar to a
base component that the conservative variant is similar to, e.g.,
an O-tRNA or O-RS, having variations in the sequence as compared to
a reference O-tRNA or O-RS. For example, an O-RS, or a conservative
variant of that O-RS, will aminoacylate a cognate O-tRNA with an
unnatural amino acid, e.g., an amino acid comprising an
N-acetylgalactosamine moiety. In this example, the O-RS and the
conservative variant O-RS do not have the same amino acid
sequences. The conservative variant can have, e.g., one variation,
two variations, three variations, four variations, or five or more
variations in sequence, as long as the conservative variant is
still complementary to the corresponding O-tRNA or O-RS.
[0047] In some embodiments, a conservative variant O-RS comprises
one or more conservative amino acid substitutions compared to the
O-RS from which it was derived. In some embodiments, a conservative
variant O-RS comprises one or more conservative amino acid
substitutions compared to the O-RS from which it was derived, and
furthermore, retains O-RS biological activity; for example, a
conservative variant O-RS that retains at least 10% of the
biological activity of the parent O-RS molecule from which it was
derived, or alternatively, at least 20%, at least 30%, or at least
40%. In some preferred embodiments, the conservative variant O-RS
retains at least 50% of the biological activity of the parent O-RS
molecule from which it was derived. The conservative amino acid
substitutions of a conservative variant O-RS can occur in any
domain of the O-RS, including the amino acid binding pocket.
[0048] Selection or screening agent: As used herein, the term
"selection or screening agent" refers to an agent that, when
present, allows for selection/screening of certain components from
a population. For example, a selection or screening agent can be,
but is not limited to, e.g., a nutrient, an antibiotic, a
wavelength of light, an antibody, an expressed polynucleotide, or
the like. The selection agent can be varied, e.g., by
concentration, intensity, etc.
[0049] In response to: As used herein, the term "in response to"
refers to the process in which an O-tRNA of the invention
recognizes a selector codon and mediates the incorporation of the
unnatural amino acid, which is coupled to the tRNA, into the
growing polypeptide chain.
[0050] Encode: As used herein, the term "encode" refers to any
process whereby the information in a polymeric macromolecule or
sequence string is used to direct the production of a second
molecule or sequence string that is different from the first
molecule or sequence string. As used herein, the term is used
broadly, and can have a variety of applications. In some aspects,
the term "encode" describes the process of semi-conservative DNA
replication, where one strand of a double-stranded DNA molecule is
used as a template to encode a newly synthesized complementary
sister strand by a DNA-dependent DNA polymerase.
[0051] In another aspect, the term "encode" refers to any process
whereby the information in one molecule is used to direct the
production of a second molecule that has a different chemical
nature from the first molecule. For example, a DNA molecule can
encode an RNA molecule (e.g., by the process of transcription
incorporating a DNA-dependent RNA polymerase enzyme). Also, an RNA
molecule can encode a polypeptide, as in the process of
translation. When used to describe the process of translation, the
term "encode" also extends to the triplet codon that encodes an
amino acid. In some aspects, an RNA molecule can encode a DNA
molecule, e.g., by the process of reverse transcription
incorporating an RNA-dependent DNA polymerase. In another aspect, a
DNA molecule can encode a polypeptide, where it is understood that
"encode" as used in that case incorporates both the processes of
transcription and translation.
[0052] Azido: As used herein, the term "azido" refers to the
chemical group --N.sub.3, typically attached to a carbon atom,
having the general structure: R--N.dbd.N.sup.+.dbd.N.sup.-
[0053] For example, the unnatural amino acid
p-azido-L-phenylalanine (FIG. 3, structure 2) comprises an azido
moiety. Also, an azido dye is a dye molecule with an azido
substituent group (see, e.g., the azido dyes 10 and 11, in FIG.
16). The term "azide" refers to a chemical compound containing the
azido group (for example, benzyl azide, sodium azide, etc.). An
aryl-azide is a aromatic molecule comprising an azide moiety, e.g.,
the unnatural amino acid p-azido-L-phenylalanine is an
aryl-azide.
[0054] Alkyne: As used herein, the term "alkyne" (also sometimes
referred to as "acetylene") refers to chemical structures
containing a triple bond between two carbon atoms, having the
general structure: C.ident.C--R where R is any atom or structure.
When used as a substituent, the alkyne moiety is termed an
"alkynyl" group. The alkynyl carbon atoms are sp.sup.2 hybridized
and form only bonds to two other atoms; one of these bonds will be
a single bond while the second bond is a triple bond. For example,
the amino acid para-propargyloxyphenylalanine (pPRO-Phe) comprises
an alkynyl group See, FIG. 15, structure 9. Because alkynyl
substituents do not appear on amino acids in nature, any alkynyl
amino acid is an unnatural amino acid. Also, FIG. 5, structure 6,
provides the chemical structure of an alkyne-derivatized
fluorescein dye.
[0055] Polypeptide: A polypeptide is any oligomer of amino acids
(natural or unnatural, or a combination thereof), of any length,
typically but not exclusively joined by covalent peptide bonds. A
polypeptide can be from any source, e.g., a naturally occurring
polypeptide, a polypeptide produced by recombinant molecular
genetic techniques, a polypeptide from a cell or translation
system, or a polypeptide produced by cell-free synthetic means. A
polypeptide is characterized by its amino acid sequence, e.g., the
primary structure of its component amino acids. As used herein, the
amino acid sequence of a polypeptide is not limited to full-length
sequences, but can be partial or complete sequences. Furthermore,
it is not intended that a polypeptide be limited by possessing or
not possessing any particular biological activity. As used herein,
the term "protein" is synonymous with polypeptide. The term
"peptide" refers to a small polypeptide, for example but not
limited to, from 2-25 amino acids in length.
[0056] Posttranslational modification: As used herein, a
posttranslational modification is a modification to a polypeptide
that can occur within a cell or in a cell free-system, either
cotranslationally or after the polypeptide has been fully
translated. Post-translational modifications can be naturally
occurring in vivo, and in many instances are required in order for
a native polypeptide to be biologically active. A wide variety of
posttranslational modifications are known to exist in vivo,
including, e.g., glycosylation and/or phosphorylation, and are
typically regulated by endogenous cellular components such as
cellular proteins. A polypeptide can be subject to multiple types
of posttranslational modifications and the modifications can be
anywhere within the polypeptide molecule.
[0057] Known posttranslational modifications include, without
limitation, acetylation, acylation, ADP-ribosylation, amidation,
covalent attachment of flavin, covalent attachment of a heme
moiety, covalent attachment of a nucleotide or nucleotide
derivative, covalent attachment of a lipid or lipid derivative,
covalent attachment of phosphotidylinositol, cross-linking,
cyclization, disulfide bond formation, demethylation, formation of
covalent cross-links, formation of cystine, formation of
pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI
anchor formation, hydroxylation, iodination, methylation,
myristoylation, oxidation, proteolytic processing, phosphorylation,
prenylation, racemization, selenoylation, sulfation, transfer-RNA
mediated addition of amino acids to proteins such as arginylation,
and ubiquitination. Such modifications are well known to those of
skill and have been described in great detail in the scientific
literature, such as, for instance, Creighton, T. E.,
Proteins--Structure And Molecular Properties, 2nd Ed., W. H.
Freeman and Company, New York (1993); Wold, F., "Posttranslational
Protein Modifications: Perspectives and Prospects," in
Posttranslational Covalent Modification of Proteins, Johnson, B.
C., ed., Academic Press, New York (1983), pp. 1-12; Seifter et al.,
"Analysis for protein modifications and nonprotein cofactors,"
Meth. Enzymol. 182:626-646 (1990), and Rattan et al., Ann. N.Y
Acad. Sci. 663:48-62 (1992).
[0058] Solid support: As used herein, the term "solid support"
refers to a matrix of material in a substantially fixed arrangement
that can be functionalized to allow synthesis, attachment or
immobilization of polypeptides (e.g., or phage comprising
polypeptides), either directly or indirectly. The term "solid
support" also encompasses terms such as "resin" or "solid phase." A
solid support can be composed of polymers, e.g., organic polymers
such as polystyrene, polyethylene, polypropylene,
polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as
co-polymers and grafts thereof. A solid support can be inorganic,
such as glass, silica, silicon, controlled-pore-glass (CPG),
reverse-phase silica, or any suitable metal. In addition to those
described herein, it is also intended that the term "solid support"
include any solid support that has received any type of coating or
any other type of secondary treatment, e.g., Langmuir-Blodgett
films, self-assembled monolayers (SAM), sol-gel, or the like.
[0059] Array: As used herein, "array" or "microarray" is an
arrangement of elements (e.g., phage-displayed polypeptides), e.g.,
present on a solid support and/or in an arrangement of vessels.
While arrays are most often thought of as physical elements with a
specified spatial-physical relationship, the present invention can
also make use of "logical" arrays, which do not have a
straightforward spatial organization. For example, a computer
system can be used to track the location of one or several
components of interest that are located in or on physically
disparate components. The computer system creates a logical array
by providing a "look-up" table of the physical location of array
members. Thus, even components in motion can be part of a logical
array, as long as the members of the array can be specified and
located. This is relevant, e.g., where the array of the invention
is present in a flowing microscale system, or when it is present in
one or more microtiter trays.
[0060] Certain array formats are sometimes referred to as a "chip"
or "biochip." An array can comprise a low-density number of
addressable locations, e.g., 2 to about 10, medium-density, e.g.,
about a hundred or more locations, or a high-density number, e.g.,
a thousand or more. Typically, the chip array format is a
geometrically-regular shape that allows for facilitated
fabrication, handling, placement, stacking, reagent introduction,
detection, and storage. It can, however, be irregular. In one
typical format, an array is configured in a row and column format,
with regular spacing between each location of member sets on the
array. Alternatively, the locations can be bundled, mixed, or
homogeneously blended for equalized treatment or sampling. An array
can comprise a plurality of addressable locations configured so
that each location is spatially addressable for high-throughput
handling, robotic delivery, masking, or sampling of reagents. An
array can also be configured to facilitate detection or
quantitation by any particular means, including but not limited to,
scanning by laser illumination, confocal or deflective light
gathering, CCD detection, and chemical luminescence. "Array"
formats, as recited herein, include but are not limited to, arrays
(i.e., an array of a multiplicity of chips), microchips,
microarrays, a microarray assembled on a single chip, arrays of
biomolecules attached to microwell plates, or any other appropriate
format for use with a system of interest.
[0061] Covalent bond: A used herein, a covalent bond is a bond
comprising shared electrons between atoms. A covalent bond is
synonymous with "chemical bond." A non-covalent bond is any bond
that is not a covalent bond. One type of non-covalent bond is an
ionic bond. An ionic bond is an attraction between oppositely
charged chemical moieties. In an ionic bond, electrons are not
shared, but rather, are unequally transferred resulting in unequal
charge distributions and positive/negative charge attractions.
BRIEF DESCRIPTION OF THE FIGURES
[0062] FIG. 1 provides examples of polynucleotide and polypeptide
sequences that find use with the invention.
[0063] FIG. 2 provides examples of amino acid sequences of
Methanococcus jannaschii tyrosyl-tRNA synthetase mutants that have
the ability to charge an orthogonal tRNA with the unnatural amino
acid para-azido-L-phenylalanine.
[0064] FIG. 3 provides the structures and corresponding names of
five (numbered 1 through 5) unnatural amino acids, which are
0-methyl-tyrosine (1), para-azido-L-phenylalanine (2),
para-acetyl-L-phenylalanine (3), para-benzoyl-L-phenylalanine (4)
and 3-(2-naphthyl)alanine (5).
[0065] FIG. 4 provides the results of an experiment demonstrating
the dependence of M13-SBP phage yields (expressed as PFU/mL) on the
presence of the corresponding unnatural amino acid.
[0066] FIG. 5 provides the chemical structure of an
alkyne-derivatized fluorescein dye (structure 6).
[0067] FIG. 6 provides a chemiluminescence image following a
Western blot analysis using anti-fluorescein (I) or anti-pIII (II)
primary antibodies, where the samples constitute reaction material
following the [3+2] cycloaddition reactions of M13KE-SBP phage,
where the phage are prepared in either the strain TTS/RS in the
presence of p-azido-L-phenylalanine 2 (a) or prepared in XL1-Blue
(b).
[0068] FIG. 7 provides the absorbance value results of a phage
streptavidin binding ELISA. Absorbance was measures at 492 nm.
[0069] FIG. 8 provides the graphical results of the phage
streptavidin binding ELISA shown in FIG. 7. (.tangle-solidup.)
M13KE; (.quadrature.) M13KE-SBP phage prepared in strain TTS/RS in
the presence of 3; (O) M13KE-SBP phage prepared in TTS/RS in the
presence of 2; (x) M13KE-SBP phage prepared in XL1-Blue.
[0070] FIG. 9 provides the results of an enrichment factor
determination of phage recovery.
[0071] FIG. 10 shows a schematic of the Staudinger conjugation
reaction involving a phage-displayed polypeptide comprising a
para-azido-L-phenylalanine residue (from phage Ph-Az) with a
phosphine 7 or 8.
[0072] FIG. 11 provides a chemiluminescence image of a Western blot
analysis of phage Ph-Az and Ph-Q after Staudinger ligation with
phosphines 7 and 8. The analysis used anti-fluorescein primary
antibody (lanes 1-4) or anti-pIII primary antibody (lanes 5-8).
[0073] FIG. 12 provides a MALDI-TOF analysis of the reaction
products from the Staudinger ligation of p-azido-L-phenylalanine 2
containing Z-domain protein with phosphine 7. Peaks A and B can be
assigned to the conjugation and reduction product, respectively;
minor peaks a.sub.2, b.sub.2, a.sub.1 and b.sub.1 are derived from
the matrix-adducts and the exclusion of methionine from A and
B.
[0074] FIG. 13 provides a MALDI-TOF spectral analysis of the
reaction products from the Staudinger ligation of pAzPhe containing
Z-domain protein with phosphine 8.
[0075] FIG. 14 provides a MALDI-TOF analysis of the reaction
products from the Staudinger ligation of p-azido-L-phenylalanine 2
containing Z-domain protein with phosphine 7 and doping with a
comparative amount of authentic p-azido-L-phenylalanine 2 Z-domain
mutant.
[0076] FIG. 15 provides the chemical structure (9) of the unnatural
alkynyl amino acid para-propargyloxyphenylalanine (pPRO-Phe; also
known as 2-amino-3-[4-(prop-2-ynyloxy)phenyl]-propionic acid
according to IUPAC nomenclature). FIG. 15 also provides the
generalized reaction chemistry of the irreversible formation of
triazole structures by a [3+2] cycloaddition reaction of an azido
and an alkyne in the presence of copper at room temperature.
[0077] FIG. 16 provides the chemical structures (10 and 11) of two
azido-functionalized dyes. Dye 10 contains a dansyl fluorophore,
and dye 11 contains a fluorescein fluorophore.
DETAILED DESCRIPTION OF THE INVENTION
[0078] There is a need for chemical reactions that modify
phage-displayed proteins in a highly selective fashion. There is
also a need for such modification reactions that can operate in
physiologically-compatible conditions in order to preserve protein
activity and phage viability. Most reactions currently used in the
art for the selective modification of proteins, e.g.,
phage-displayed proteins, involve covalent bond formation between
nucleophilic and electrophilic reaction partners that target
naturally occurring nucleophilic residues in the amino acid side
chains. Selectivity in these cases is determined by the number and
accessibility of the nucleophilic residues in the protein.
Unfortunately, naturally occurring proteins frequently contain
poorly positioned (e.g., inaccessible) reaction sites or multiple
reaction targets (e.g., lysine, histidine and cysteine residues),
resulting in poor selectivity in the modification reactions, making
highly targeted protein modification by nucleophilic/electrophilic
reagents difficult. Furthermore, the sites of modification are
typically limited to the naturally occurring nucleophilic side
chains of lysine, histidine or cysteine. Modification at other
sites is difficult or impossible.
[0079] The present invention provides solutions to these problems.
The invention provides systems for the programmed, site-specific
biosynthetic incorporation of unnatural amino acids with novel
properties into phage-displayed proteins by manipulating orthogonal
translation systems to work in conjunction with recombinant phage
expression reagents. The invention provides methods for the
subsequent targeted modification of those unnatural amino acid
residues that are incorporated into phage-displayed polypeptides.
We describe herein novel compositions (e.g., phage comprising
various posttranslational modifications) and novel methods for the
generation of post-translationally modified phage.
[0080] The phage-production systems provided by the present
invention take advantage of orthogonal translation systems that use
E. coli host cells for the selective incorporation of unnatural
amino acids into phage-displayed polypeptides, and the subsequent
modification of those polypeptides using selective modification of
the unnatural amino acid residue. Various chemistries for the
modification of the unnatural amino acid residue in the
phage-displayed polypeptide are contemplated and demonstrated
herein, including [3+2] cycloaddition reactions and Staudinger
ligations.
[0081] The orthogonal translation systems finding use with the
invention comprise an orthogonal tRNA that recognizes a selector
codon and an orthogonal aminoacyl-tRNA synthetase that specifically
charges the orthogonal tRNA with an unnatural amino acid in E. coli
host cells. The incorporation of the unnatural amino acid into the
phage-displayed protein of interest can be programmed to occur at
any desired position by engineering the polynucleotide encoding the
protein of interest to contain the selector codon at the desired
site, thereby signaling the incorporation of the unnatural amino
acid.
[0082] The present disclosure describes the incorporation of a
number of unnatural amino acids into phage displayed polypeptides.
These amino acids include O-methyl-tyrosine,
p-azido-L-phenylalanine, p-acetyl-L-phenylalanine,
p-benzoyl-L-phenylalanine and 3-(2-naphthyl)alanine. Aryl-azide
amino acids, e.g., para-azido-L-phenylalanine, present attractive
targets for specific and regioselective posttranslational
modifications. Unnatural amino acids comprising alkynyl-groups,
e.g., para-propargyloxyphenylalanine, are also contemplated for use
in phage-displayed polypeptides as targets for posttranslational
modification. In some embodiments of the invention, the
posttranslational modification of the unnatural amino acid in the
phage-displayed polypeptide is done using relatively mild and
physiologically-compatible in vitro or in vivo reaction conditions
that preserve phage viability.
[0083] Because of the unique reaction chemistries of aryl-azide and
alkynyl unnatural amino acids, phage-displayed proteins into which
they are incorporated can be modified with extremely high
selectivity. In some cases, the unnatural amino acid reactive group
has the advantage of being completely alien to in vivo systems,
thereby improving reaction selectivity.
[0084] The nature of the material that is conjugated to a
phage-displayed protein via an unnatural amino acid target is not
particularly limited and can be any desired entity, e.g., dyes,
fluorophores, crosslinking agents, saccharide derivatives, polymers
(e.g., derivatives of polyethylene glycol), photocrosslinkers,
cytotoxic compounds, affinity labels, derivatives of biotin,
resins, beads, a second protein or polypeptide (or more),
polynucleotide(s) (e.g., DNA, RNA, etc.), metal chelators,
cofactors, fatty acids, carbohydrates, and the like. The disclosure
herein describes the experimental use of derivatized fluorescein or
dansyl fluorophore dyes as conjugated material. However, it is not
intended that the invention be limited to the use of these
conjugated materials, as a wide range of conjugatable materials is
contemplated, e.g., those listed above.
Phage Display
[0085] Phage display technology has become a widely used technique
in diverse biological disciplines. Phage display has found
particular use in peptide (i.e., polypeptide) library screening
protocols. Various applications include affinity selection (e.g.,
target receptor selection), epitope mapping and mimicking,
identification of new receptors and natural ligands, drug
discovery, epitope discovery for vaccine development and
diagnostics, and study of DNA-binding proteins. A variety of
resources are available that describe the many protocols, reagents
and variant phage genomes (and variant phage genes) that find use
in phage-display technology. See, e.g., Smith and Petrenko, Chem.
Rev., 97:391-410 (1997); Sidhu, Bimolecular Engineering 18:57-63
(2001); Rodi and Makowski, Current Opinion in Biotechnology
10:87-93 (1999); and Willats, Plant Molecular Biology 50:837-854
(2002).
[0086] Experiments described in the present disclosure use the
filamentous M13KE phage system (New England BioLabs, Inc.). M13KE
is a derivative of M13mp19 designed for expression of peptides as
N-terminal pIII fusions in phage display applications (Zwick et al.
(1998) Anal. Biochem., 264:87-97). Libraries constructed in M13KE
are pentavalent (i.e., all five copies of pIII in the mature virion
carry the fused peptide). Relative to the parent M13mp19, Acc65
I/Kpn I and Eag I sites have been introduced flanking the pIII
leader peptidase cleavage site, and the Acc65 I/Kpn I site in the
multiple cloning site (MCS) was deleted. Phage displayed random
peptide libraries are constructed by annealing an extension primer
to a synthetic oligonucleotide encoding the random peptide library
and a portion of the pIII leader sequence, extending with DNA
polymerase, and digesting with Acc65 I and Eag I (Noren and Noren
(2001) Methods 23:169-178). The resulting cleaved duplex is
inserted into M13KE which has been digested with the same
enzymes.
[0087] Although the Examples provided herein use the M13KE phage
system, it is not intended that the invention be limited to that
particular system. Indeed, one of skill in the art recognizes
alternative phage display reagents and protocols that are available
and also find use with the compositions and methods of the
invention. These alternative reagents and protocols do not depart
from the scope of the invention, and are encompassed by the claimed
invention.
[0088] Generally, display of a polypeptide of interest is
accomplished by fusing the polypeptide with a phage capsid (coat)
protein, or a fragment, mutant or other variant of a capsid
protein. These capsid proteins can include pIII, pVI, pVII, pVIII
and pIX. For the purpose of demonstrating (but not limiting) the
invention, the Examples herein describe the generation of
phage-displayed fusion polypeptides comprising the phage pIII coat
protein amino acid sequence. It is not intended that the invention
be limited to use of the pIII polypeptide sequence for the display
of the fused protein of interest.
[0089] In some phage systems, a linker protease recognition signal
sequence can be engineered into the cased fusion polypeptide,
thereby facilitating cleavage and/or release of the fused protein
moiety. A wide variety of protease signal sequences are known,
including but not limited to Factor Xa, Factor XIa, Kallikvein,
thrombin, Factor XIIa, collagenase and enterokinase. Any suitable
protease recognition signal and corresponding protease can be used
with the present invention.
[0090] Similarly, to demonstrate (but not to limit) the present
invention, the disclosure herein demonstrates that an unnatural
amino acid moiety can be incorporated into a model phage-displayed
fusion protein comprising the streptavidin binding peptide (SBP),
which is then post-translationally modified. It is not intended
that the incorporation of an unnatural amino acid be limited to
such a model protein. From the present disclosure, it will be clear
that the incorporation of an unnatural amino acid into any given
phage-displayed protein of interest is advantageous for a wide
variety of proteins for use in therapeutic and research
purposes.
[0091] The invention also provides phage comprising polypeptides
comprising at least one unnatural amino acid that is
post-translationally modified, where the phage is purified or
isolated. For example, the phage can be purified and/or isolated by
PEG precipitation and/or centrifugation. See, Example 3. Additional
phage precipitation and purification/isolation techniques are also
known in the art, for example, using affinity purification schemes
such as immuno-affinity.
Phage Display Libraries and Arrays
[0092] The phage display of polypeptides comprising unnatural amino
acids that are post-translationally modified finds a variety of
uses. Discussion of the uses of phage displayed polypeptides is
found in a variety of sources, for example, Smith and Petrenko,
Chem. Rev., 97:391-410 (1997).
[0093] In some embodiments, the phage-displayed polypeptide
comprising an unnatural amino acid that is post-translationally
modified is a member of a plurality of phage carrying the same or
different encoded polypeptides, or variants of the same
polypeptides (all comprising at least one unnatural amino acid). In
some embodiments, where the phage display different polypeptide
sequences, the displayed polypeptides comprise a library. for
example, a randomized mutant library of a coding sequence of
interest.
[0094] Where the phage-displayed polypeptides comprising an
unnatural amino acid that is post-translationally modified
constitute a library, there is generally a selection step that is
applied to select the desired polypeptide species (or the nucleic
acid encoding that polypeptide species) from the pool of displayed
polypeptide candidates. Selection can consist of culling an initial
population of phage-borne polypeptides to give a subpopulation with
increased "fitness" according to some user-defined criterion. In
most cases, the library input to a first round of selection is a
very large initial number, and the selected subpopulation is a
fraction of the initial population, where fitter clones are over
represented. This population can be "amplified" by infecting fresh
bacterial host cells, so that each individual phage in the
subpopulation is amplified in the new amplified stock. The
amplified population can then be subjected to further rounds of
selection to obtain an ever-fitter subset of the starting
peptides.
[0095] Generally, there are two pivotal parameters of selection,
which can often be manipulated to some extent in order to enhance
the efficacy of selection. First, stringency is the degree to which
polypeptides with higher fitness are favored over peptides with
lower fitness; second, yield is the fraction of particles with a
given fitness that survive selection. The ultimate goal of
selection is usually to isolate peptides with the best fitness.
However, selection for the most fit polypeptides must be balanced
with an appropriate stringency to allow reasonable yield. If
stringency is set too high, the yield of a specifically selected
phage will fall below the background of a nonspecifically isolated
phage, and the power to discriminate in favor of high fitness is
lost.
[0096] One of the most common selection pressures imposed on
phage-displayed polypeptide populations is affinity for a target
receptor. Affinity selection is ordinarily accomplished by minor
modifications of standard affinity purification techniques in
common use in biochemistry. Thus e.g., a receptor is tethered to a
solid support, and the phage mixture is passed over the immobilized
receptor. A small minority of the phage-displayed polypeptides in
the library bind the receptor, and are captured on the surface or
matrix, allowing unbound phages to be washed away. Finally, the
bound phages are eluted in a solution that loosens the
receptor-peptide interaction, yielding an "eluate" population of
phages that is enriched for receptor-binding clones. The eluted
phages are still infective and are propagated simply by infecting
fresh bacterial host cells, yielding an "amplified" eluate that can
serve as input to another round of affinity selection. Phage clones
from the final eluate are propagated and characterized
individually. The amino acid sequences of the peptides responsible
for binding the target receptor are determined simply by
ascertaining the corresponding coding sequence in the viral
DNA.
[0097] Phage-borne polypeptides can be selected on the basis of
fitness criteria other than affinity for a target receptor. For
example, the phage carrying displayed polypeptide libraries can be
selected based on a desired biological activity (e.g., an enzymatic
activity, an improved enzymatic activity, or an activity that
displays resistance to certain agents or repressors.).
[0098] Variations of these phage-selection schemes are numerous and
are known to one of skill in the art. Furthermore, numerous
publications are devoted to the subject of phage library screening
methodologies.
[0099] In some embodiments, the phage displaying the polypeptide
comprising the unnatural amino acid can be immobilized on a
suitable support. In this aspect, the unnatural amino acid that is
incorporated into the phage-displayed polypeptide can be optionally
used as a reactive moiety to form a coupling with the immobilized
phase, e.g., using a [3+2] cycloaddition reaction or a Staudinger
ligation reaction.
[0100] The nature of the solid support to which a phage (or phage
library) can be immobilized is not limited. For example, phage can
be affixed to solid supports that include polystyrene dishes,
impermeable plastic beads, nylon or nitrocellulose membranes,
paramagnetic beads and permeable beaded agarose gels. In some
embodiments, the immobilized phage are arranged in some specified
relationship, i.e., they form an array. Discussion of using
unnatural amino acids to form linkages with solid supports, as well
as solid support formats and array can be found, for example, in
International Publication WO 2004/058946, entitled "PROTEIN
ARRAYS."
Orthogonal Translation System Components
[0101] In some aspects of the invention, the unnatural amino acid
p-azido-L-phenylalanine (see FIG. 3, structure 2) is incorporated
into a phage-displayed polypeptide of interest. When incorporated
into a phage-displayed polypeptide, this unnatural amino acid can
serve as chemical target for [3+2] cycloaddition reactions and in
Staudinger modification reactions for posttranslational
modification of the phage displayed polypeptide.
[0102] Orthogonal components for the incorporation of this
unnatural amino acid are provided herein. FIG. 1 provides seven
mutant Methanococcus janaschii tyrosyl-tRNA synthetase species (see
SEQ ID NOS: 4 through 10) that charge an orthogonal suppressor tRNA
with p-azido-L-phenylalanine, subsequently resulting in the
incorporation of the p-azido-L-phenylalanine during translation in
response to a selector codon. An orthogonal suppressor tRNA finding
use with the invention is provided in SEQ ID NO: 1.
[0103] Suitable orthogonal tRNAs and aminoacyl-tRNA synthetases for
the incorporation of p-azido-L-phenylalanine are also described in
Chin et al., J. Am. Chem. Soc., (2002) 124:9026-9027; and
International Publications WO 2002/086075, entitled "METHODS AND
COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA
SYNTHETASE PAIRS;" WO 2002/085923, entitled "IN VIVO INCORPORATION
OF UNNATURAL AMINO ACIDS;" each of which are hereby incorporated by
reference in their entirety for all purposes. In addition, the
prior art and present disclosure also provide guidance for the
synthesis of additional orthogonal tRNAs and orthogonal aminoacyl
tRNA synthetases that are not specifically recited by sequence.
[0104] In other aspects of the invention, an unnatural alkynyl
amino acid, is incorporated into a phage-displayed polypeptide of
interest, also to serve as a target for posttranslational
modification. For example, the unnatural alkynyl amino acid
para-propargyloxyphenylalanine (pPRO-Phe; see structure 9 in FIG.
15) finds use for this purpose. An alkynyl amino acid can serve as
a target for [3+2] cycloaddition reactions. Orthogonal components
for the incorporation of this unnatural amino acid are provided in,
for example, Deiters et al, Bioorganic & Medicinal Chemistry
Letters 15:1521-1524 (2005) and International Publication No.
WO2006/034332, filed on Sep. 20, 2005.
[3+2] Cycloadditon Reaction
[0105] Unnatural amino acid side chains (e.g., on an aryl-azide
amino acid or an alkynyl amino acid) can be incorporated into a
phage-displayed protein of interest, then specifically and
regioselectively modified by a Huisgen [3+2] cycloaddition reaction
(see, Padwa, In Comprehensive Organic Synthesis; [Trost, B. M.,
Ed.] Pergamon: Oxford, 1991, Vol. 4, p 1069-1109; Huisgen, In
1,3-Dipolar Cycloaddition Chemistry, [Padwa, A., Ed.] Wiley: New
York, 1984; p 1-176). The general reaction chemistry of the [3+2]
cycloaddition reaction is shown in FIG. 15, where an azide moiety
reacts with the alkynyl moiety. This reaction is irreversible and
results in the formation of a triazole linkage.
[0106] As shown in FIG. 15, the R groups that are associated with
either the azido or alkynyl substituents in the [3+2] cycloaddition
reaction is not particularly limiting. In some aspects, the azido
group forms part of an aryl-azide unnatural amino acid, for
example, p-azido-L-phenylalanine, that is incorporated into a
phage-displayed polypeptide (see, Example 4). In that
configuration, the alkynyl moiety is attached to a reagent (e.g.,
the alkynyl-derivatized fluorescein dye shown in FIG. 5, structure
6 that can then be reacted with the phage displayed polypeptide,
resulting in a post-translationally modified phage. The nature of
the R group associated with the alkynyl group is not particularly
limited.
[0107] A reverse configuration for the [3+2] cycloaddition reaction
can also be employed. In this scenario, the alkynyl group is part
of an alkynyl unnatural amino acid, for example,
para-propargyloxyphenylalanine (see, FIG. 15, structure 9), that is
incorporated into a phage-displayed polypeptide. In this
configuration, the azido moiety is attached to a reagent (e.g., the
azido-derivatized dansyl and fluorescein dyes shown in FIG. 16,
structures 10 and 11) that can then be reacted with the phage
displayed polypeptide, resulting in a post-translationally modified
phage. The nature of the R group associated with the azido group is
not particularly limited.
[0108] The chemistries of alkynyl and azido groups have the
advantage of being completely alien to the chemistries of the
endogenous functional groups present in proteins in vivo. When the
[3+2] cycloaddition reaction is conducted in the presence of
copper(I) at room temperature in aqueous media (conditions mild
enough for modifying biological samples), it proceeds in a
completely regioselective fashion (Rostovtsev et al. (2002) Angew.
Chem. Int. Ed., 41:2596) and can be used to selectively modify
phage-displayed proteins into which alkynyl or azido functional
groups have been introduced, e.g., by use of orthogonal translation
system (Deiters et al. (2003) J. Am. Chem. Soc., 125:11782; Wang et
al. (2003) J. Am. Chem. Soc., 125:3192; Link and Tirrell (2003) J.
Am. Chem. Soc., 125:11164). Because this method involves a
cycloaddition rather than a nucleophilic substitution, proteins can
be modified with extremely high selectivity. This reaction has the
benefits that it can be carried out at room temperature under
aqueous conditions with excellent regioselectivity (1,4>1,5) by
the addition of catalytic amounts of Cu(I) salts to the reaction
mixture (Tornoe et al., (2002) J. Org. Chem., 67:3057-3064;
Rostovtsev et al., (2002) Angew. Chem., Int. Ed.,
41:2596-2599).
[0109] For the purpose of demonstrating (but not limiting) the
invention, the Examples herein describe the use of dansyl and
fluorescein dyes that have been derivatized with either azido or
alkynyl moieties and can be used in the [3+2] cycloaddition
reaction. However, as it should be clear to one of skill in the
art, it is not intended that the invention be limited to use of
these derivatized dyes in the [3+2] cycloaddition reaction. Indeed,
this chemistry permits the posttranslational modification of the
phage-displayed polypeptide (and as a result, the posttranslational
modification of the phage) with any molecule that can be
derivatized with an azido or alkynyl moiety. It is well within the
means of one of skill in the art to synthesize an azido or alkynyl
derivative of any particular molecule of interest. For example,
many texts and protocols are available describing how to synthesize
azido compounds. For a general reference see: Patai, Saul, "The
chemistry of the azido group" in The Chemistry of Functional
Groups, London, N.Y., Interscience Publishers, 1971.
[0110] In other aspects, the invention provides compositions and
methods for the generation of PEGylated phage-displayed
polypeptides by using azido derivatives of polyethylene glycol
(azido-PEG) for use in [3+2] cycloaddition conjugation reactions
with alkynyl-containing phage-displayed polypeptides. The
generalized structure of an azido polyethylene glycol is:
N.sub.3--CH.sub.2--(CH.sub.2--O--CH.sub.2)n--CH.sub.2OR where R is
H or CH.sub.3, and where n is an integer between, e.g., 50 and
10,000, 75 and 5,000, 100 and 2,000, 100 and 1,000, etc. In various
embodiments of the invention, the azido polyethylene glycol has a
molecular weight of, e.g., about 5,000 to about 100,000 Da (i.e.,
about 5 kDa to about 100 kDa), about 20,000 to about 50, 000 Da,
about 20,000 to about 10,000 Da (e.g., 20,000 Da), etc. Techniques
for the synthesis of an azido polyethylene glycol are well known to
one of skill in the art. For example a polyethylene glycol molecule
containing an electrophilic group (e.g., a bromide or an
N-hydroxysuccinimide ester) can be reacted with a nucleophilic
molecule containing an azido group (e.g., sodium azide or
3-azidopropylamine) to generate an azido polyethylene glycol.
[0111] Azido-PEG finds use with the invention when bioconjugated to
an alkynyl-containing phage-displayed protein via a triazole
linkage. Derivatization of protein-based therapeutics with
polyethylene glycol (PEGylation) can often improve pharmacokinetic
and pharmacodynamic properties of the proteins and thereby, improve
efficacy and minimize dosing frequency. The various advantages of
PEGylation of protein therapeutics are discussed and illustrated
in, for example, Deiters et al., "Site-specific PEGylation of
proteins containing unnatural amino acids," Bioorganic &
Medicinal Chemistry Letters 14:5743-5745 (2004).
[0112] In addition, other advantages associated with the generation
of phage-displayed polypeptides comprising unnatural alkynyl amino
acids that also contain an ester linkage are contemplated. For
example, a PEGylated polypeptide created by using an alkynyl amino
acid with an ester linkage can allow the slow release of the
polypeptide by saponification of the ester linkages in vivo or in
vitro. Also, using a polymeric support (an azido resin) in place of
a azido-PEG molecule enables a protein affinity purification. The
triazole covalent linkage permits very strong washing steps, and
the use of the ester alkynyl amino acid allows release of the
phage-displayed protein by treatment with a base. Significantly,
such an affinity purification scheme no longer requires the
presence of an artificial tag (e.g., hexahistidine) or epitope on
the protein of interest for the purification. Depending on the
unnatural amino acid used, an essentially wild-type (native)
polypeptide can be released from the affinity resin following the
cleavage step.
[0113] Unnatural alkynyl amino acids with ester linkages can by
synthesized and incorporated into proteins. See, for example, the
ester linkage alkynyl amino acids in International Publication No.
WO2006/034332, filed on Sep. 20, 2005. After bioconjugation via
[3+2] cycloaddition, the ester linkages could be cleaved by
saponification in vivo or in vitro; an application would be, e.g.,
the slow release of the peptide part from a PEGylated
phage-displayed protein.
[0114] In other aspects, the invention provides compositions and
methods for the generation of PEGylated phage-displayed
polypeptides by using alkynyl derivatives of polyethylene glycol
(alkynyl-PEG) for use in [3+2] cycloaddition conjugation reactions
with azido-containing unnatual amino acids that are incorporated
into phage-displayed polypeptides.
Staudinger Reaction
[0115] The Staudinger ligation has been previously used to
selectively modify cell surface carbohydrates in both cellular and
in vivo systems (Saxon and Bertozzi, Science 2000, 287, 2007-2010;
Prescher et al., Nature 2004, 430, 873-877). The reaction proceeds
in excellent yields under aqueous conditions and is highly
selective for azide moieties. The Staudinger ligation has also been
used to selectively modify proteins that contain azidohomoalanine
substituted for methionine residues (Kiick et al., Proc. Natl.
Acad. Sci. U.S.A. 2002, 101, 7566-7571). However, the selectivity
of this Staudinger ligation approach is intrinsically limited since
each methionine residue in a proteins as well as in the entire
proteome are substituted with azidohomoalanine, often in
competition with the native amino acid.
[0116] The invention provides methods for producing a
post-translationally modified phage, where the phage comprises a
displayed polypeptide comprising an aryl-azide unnatural amino
acid, e.g., p-azido-L-phenylalanine. That unnatural amino acid is
then efficiently and specifically modified using a Staudinger
ligation reaction to produce a post-translationally modified phage.
FIG. 10 shows a schematic of the Staudinger ligation reaction of a
phage-displayed polypeptide comprising a p-azido-L-phenylalanine
residue (from phage Ph-Az) with two different spectroscopic probe
phosphine molecules (structures 7 and 8). This methodology is
explained in detail in Example 6.
[0117] For the purpose of demonstrating (but not limiting) the
invention, the Examples herein describe the use of two
spectroscopic probes that have been suitably derivatized for use in
the Staudinger ligation reaction. However, as it should be clear to
one of skill in the art, it is not intended that the invention be
limited to use of these derivatized phosphine molecules in the
Staudinger reaction. The Staudinger reaction chemistry permits the
posttranslational modification of the phage-displayed polypeptide
(and as a result, the posttranslational modification of the phage)
with any molecule that can be suitably derivatized. It is well
within the means of one of skill in the art to synthesize suitable
derivatives of any particular molecule of interest for use in the
conjugation.
Orthogonal tRNA/AMINOACYL-tRNA Synthetase Technology
[0118] An understanding of the novel compositions and methods of
the present invention is facilitated by an understanding of the
activities associated with orthogonal tRNA and orthogonal
aminoacyl-tRNA synthetase pairs. Discussions of orthogonal tRNA and
aminoacyl-tRNA synthetase technologies can be found, for example,
in International Publications WO 2002/085923, WO 2002/086075, WO
204/09459, WO 2005/019415, WO 2005/007870 and WO 2005/007624. See
also, Wang and Schultz "Expanding the Genetic Code," Angewandte
Chemie Int. Ed., 44(l):34-66 (2005), the content of which is
incorporated by reference in its entirety.
[0119] In order to add additional reactive unnatural amino acids to
the genetic code, new orthogonal pairs comprising an aminoacyl-tRNA
synthetase and a suitable tRNA are needed that can function
efficiently in the host translational machinery, but that are
"orthogonal" to the translation system at issue, meaning that it
functions independently of the synthetases and tRNAs endogenous to
the translation system. Desired characteristics of the orthologous
pair include tRNA that decode or recognize only a specific codon,
e.g., a selector codon, that is not decoded by any endogenous tRNA,
and aminoacyl-tRNA synthetases that preferentially aminoacylate (or
"charge") its cognate tRNA with only one specific unnatural amino
acid. The O-tRNA is also not typically aminoacylated by endogenous
synthetases. For example, in E. coli, an orthogonal pair will
include an aminoacyl-tRNA synthetase that does not cross-react with
any of the endogenous tRNA, e.g., which there are 40 in E. coli,
and an orthogonal tRNA that is not aminoacylated by any of the
endogenous synthetases, e.g., of which there are 21 in E. coli.
[0120] The invention provides phage-displayed polypeptides
comprising unnatural amino acids, where the unnatural amino acid
(and consequently the phage) are post-translationally modified. The
incorporation of the unnatural amino acid into the phage-displayed
protein is accomplished by adapting orthogonal pairs for the
genetic encoding of unnatural amino acids into proteins in E. coli,
where the orthogonal components do not cross-react with endogenous
E. coli components of the translational machinery of the host cell,
but recognize the desired unnatural amino acid and incorporate it
into proteins in response to the selector codon (e.g., an amber
nonsense codon, TAG). The orthogonal components provided by the
invention include orthogonal aminoacyl-tRNA synthetases derived
from Methanococcus jannaschii tyrosyl tRNA-synthetase, and the
mutant tyrosyl tRNA.sub.CUA amber suppressor, which function as an
orthogonal pair in a eubacterial host cell. In this system, the
mutant aminoacyl-tRNA synthetases aminoacylate the suppressor tRNA
with its respective unnatural amino acid and not with any of the
common twenty amino acids.
[0121] This invention provides phage-displayed polypeptides
comprising unnatural amino acids, where the unnatural amino acid
(and consequently the phage) are post-translationally modified, and
methods for producing same. These methods utilize orthogonal
tRNA-aminoacyl-tRNA synthetase pairs, e.g., O-tRNA/ O-RS pairs that
can be used to incorporate the unnatural amino acid into the
phage-displayed protein. An O-tRNA/O-RS pair is capable of
mediating incorporation of an unnatural amino acid, for example, an
unnatural amino acid shown in FIG. 3 or FIG. 15, into a protein
that is encoded by a polynucleotide, where the polynucleotide
comprises a selector codon that is recognized by the O-tRNA, e.g.,
in vivo. The anticodon loop of the O-tRNA recognizes the selector
codon on an mRNA and incorporates its unnatural amino acid at this
site in the polypeptide. Generally, an orthogonal aminoacyl-tRNA
synthetase preferentially aminoacylates (or charges) its O-tRNA
with only one specific unnatural amino acid.
[0122] The ability to incorporate an unnatural amino acid
site-specifically into phage-displayed proteins can facilitate the
study of proteins by enabling the post-translational modification
of those proteins, as well as enable the engineering of proteins
with novel properties. For example, expression of proteins
containing one or more unnatural amino acids can facilitate the
study of proteins by specific labeling, alter catalytic function of
enzymes, improve biological activity or reduce cross-reactivity to
a substrate, crosslink a protein with other proteins, small
molecules or biomolecules, reduce or eliminate protein degradation,
improve half-life of proteins in vivo (e.g., by pegylation or other
modifications of introduced reactive sites), etc.
Orthogonal tRNA/Orthogonal AMINOACYL-tRNA Synthetases and Pairs
Thereof
[0123] Orthogonal translation systems that are suitable for making
proteins that include one or more unnatural amino acid are
generally described in, for example, International Publication
Numbers WO 2002/086075, entitled "METHODS AND COMPOSITION FOR THE
PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;" WO
2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO
ACIDS;" and WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC
GENETIC CODE;" WO 2005/019415, filed Jul. 7, 2004; WO 2005/007870,
filed Jul. 7, 2004 and WO 2005/007624, filed Jul. 7, 2004. Each of
these applications is incorporated herein by reference in its
entirety. See also, Wang and Schultz "Expanding the Genetic Code,"
Angewandte Chemie Int. Ed., 44(1):34-66 (2005); Deiters et al,
Bioorganic & Medicinal Chemistry Letters 15:1521-1524 (2005);
Chin et al., J. Am. Chem. Soc. 2002, 124, 9026-9027; and
International Publication No. WO2006/034332, filed on Sep. 20,
2005, the contents of each of which are incorporated by reference
in their entirety.
[0124] Such translation systems generally comprise cells (which can
be non-eukaryotic cells such as E. coli, or eukaryotic cells such
as yeast) that include an orthogonal tRNA (O-tRNA), an orthogonal
aminoacyl tRNA synthetase (O-RS), and an unnatural amino acid,
where the O-RS aminoacylates the O-tRNA with the unnatural amino
acid. An orthogonal pair of the invention includes an O-tRNA, e.g.,
a suppressor tRNA, a frameshift tRNA, or the like, and an O-RS.
Individual components are also provided in the invention.
[0125] In general, when an orthogonal pair recognizes a selector
codon and loads an amino acid in response to the selector codon,
the orthogonal pair is said to "suppress" the selector codon. That
is, a selector codon that is not recognized by the translation
system's (e.g., the cell's) endogenous machinery is not ordinarily
translated, which can result in blocking production of a
polypeptide that would otherwise be translated from the nucleic
acid. An O-tRNA of the invention recognizes a selector codon and
includes at least about, e.g., a 45%, a 50%, a 60%, a 75%, a 80%,
or a 90% or more suppression efficiency in the presence of a
cognate synthetase in response to a selector codon as compared to
the suppression efficiency of an O-tRNA comprising or encoded by a
polynucleotide sequence as set forth in the sequence listing
herein. The O-RS aminoacylates the O-tRNA with an unnatural amino
acid of interest. The cell uses the O-tRNA/ O-RS pair to
incorporate the unnatural amino acid into a growing polypeptide
chain, e.g., via a nucleic acid that comprises a polynucleotide
that encodes a polypeptide of interest, where the polynucleotide
comprises a selector codon that is recognized by the O-tRNA. In
certain desirable aspects, the cell can include an additional
O-tRNA/ O-RS pair, where the additional O-tRNA is loaded by the
additional O-RS with a different unnatural amino acid. For example,
one of the O-tRNAs can recognize a four base codon and the other
can recognize a stop codon. Alternately, multiple different stop
codons or multiple different four base codons can specifically
recognize different selector codons.
[0126] In certain embodiments of the invention, a cell such as an
E. coli cell or a yeast cell that includes an orthogonal tRNA
(O-tRNA), an orthogonal aminoacyl-tRNA synthetase (O-RS), an
unnatural amino acid and a nucleic acid that comprises a
polynucleotide that encodes a polypeptide of interest (e.g., the
phage-displayed fusion polypeptide), where the polynucleotide
comprises the selector codon that is recognized by the O-tRNA. The
translation system can also be a cell-free system, e.g., any of a
variety of commercially available "in vitro"
transcription/translation systems in combination with an O-tRNA/ORS
pair and an unnatural amino acid as described herein.
[0127] In one embodiment, the suppression efficiency of the O-RS
and the O-tRNA together is about, e.g., 5 fold, 10 fold, 15 fold,
20 fold, or 25 fold or more greater than the suppression efficiency
of the O-tRNA lacking the O-RS. In some aspect, the suppression
efficiency of the O-RS and the O-tRNA together is at least about,
e.g., 35%, 40%, 45%, 50%, 60%, 75%, 80%, or 90% or more of the
suppression efficiency of an orthogonal synthetase pair as set
forth in the sequence listings herein.
[0128] As noted, the invention optionally includes multiple
O-tRNA/O-RS pairs in a cell or other translation system, which
allows incorporation of more than one unnatural amino acid into a
phage-displayed polypeptide. For example, the cell can further
include an additional different O-tRNA/O-RS pair and a second
unnatural amino acid, where this additional O-tRNA recognizes a
second selector codon and this additional O-RS preferentially
aminoacylates the O-tRNA with the second unnatural amino acid. For
example, a cell that includes an O-tRNA/O-RS pair (where the O-tRNA
recognizes, e.g., an amber selector codon), can further comprise a
second orthogonal pair, where the second O-tRNA recognizes a
different selector codon, e.g., an opal codon, a four-base codon,
or the like. Desirably, the different orthogonal pairs are derived
from different sources, which can facilitate recognition of
different selector codons.
[0129] The O-tRNA and/or the O-RS can be naturally occurring or can
be, e.g., derived by mutation of a naturally occurring tRNA and/or
RS, e.g., by generating libraries of tRNAs and/or libraries of RSs,
from any of a variety of organisms and/or by using any of a variety
of available mutation strategies. For example, one strategy for
producing an orthogonal tRNA/ aminoacyl-tRNA synthetase pair
involves importing a heterologous (to the host cell)
tRNA/synthetase pair from, e.g., a source other than the host cell,
or multiple sources, into the host cell. The properties of the
heterologous synthetase candidate include, e.g., that it does not
charge any host cell tRNA, and the properties of the heterologous
tRNA candidate include, e.g., that it is not aminoacylated by any
host cell synthetase. In addition, the heterologous tRNA is
orthogonal to all host cell synthetases.
[0130] A second strategy for generating an orthogonal pair involves
generating mutant libraries from which to screen and/or select an
O-tRNA or O-RS. These strategies can also be combined.
Orthogonal tRNA (O-tRNA)
[0131] An orthogonal tRNA (O-tRNA) desirably mediates incorporation
of an unnatural amino acid into a protein that is encoded by a
polynucleotide that comprises a selector codon that is recognized
by the O-tRNA, e.g., in vivo or in vitro. In certain embodiments,
an O-tRNA of the invention includes at least about, e.g., a 45%, a
50%, a 60%, a 75%, a 80%, or a 90% or more suppression efficiency
in the presence of a cognate synthetase in response to a selector
codon as compared to an O-tRNA comprising or encoded by a
polynucleotide sequence as set forth in the O-tRNA sequences in the
sequence listing herein.
[0132] Suppression efficiency can be determined by any of a number
of assays known in the art. For example, a .beta.galactosidase
reporter assay can be used, e.g., a derivatized lacZ plasmid (where
the construct has a selector codon n the lacZ nucleic acid
sequence) is introduced into cells from an appropriate organism
(e.g., an organism where the orthogonal components can be used)
along with plasmid comprising an O-tRNA of the invention. A cognate
synthetase can also be introduced (either as a polypeptide or a
polynucleotide that encodes the cognate synthetase when expressed).
The cells are grown in media to a desired density, e.g., to an
OD.sub.600 of about 0.5, and .beta.-galactosidase assays are
performed, e.g., using the BetaFluor.TM. .beta.-Galactosidase Assay
Kit (Novagen). Percent suppression can be calculated as the
percentage of activity for a sample relative to a comparable
control, e.g., the value observed from the derivatized lacZ
construct, where the construct has a corresponding sense codon at
desired position rather than a selector codon.
[0133] Examples of O-tRNAs of the invention are set forth in the
sequence listing herein. See also, the tables, examples and figures
herein for sequences of exemplary O-tRNA and O-RS molecules. See
also, the section entitled "Nucleic Acid and Polypeptide Sequence
and Variants" herein. In an RNA molecule, such as an O-RS MRNA, or
O-tRNA molecule, Thymine (T) is replace with Uracil (U) relative to
a given sequence (or vice versa for a coding DNA), or complement
thereof. Additional modifications to the bases can also be
present.
[0134] The invention also includes conservative variations of
O-tRNAs corresponding to particular O-tRNAs herein. For example,
conservative variations of O-tRNA include those molecules that
function like the particular O-tRNAs, e.g., as in the sequence
listing herein and that maintain the tRNA L-shaped structure by
virtue of appropriate self-complementarity, but that do not have a
sequence identical to those, e.g., in the sequence listing, figures
or examples herein (and, desirably, are other than wild type tRNA
molecules). See also, the section herein entitled "Nucleic acids
and Polypeptides Sequence and Variants."
[0135] The composition comprising an O-tRNA can further include an
orthogonal aminoacyl-tRNA synthetase (O-RS), where the O-RS
preferentially aminoacylates the O-tRNA with an unnatural amino
acid. In certain embodiments, a composition including an O-tRNA can
further include a translation system (e.g., in vitro or in vivo). A
nucleic acid that comprises a polynucleotide that encodes a
polypeptide of interest, where the polynucleotide comprises a
selector codon that is recognized by the O-tRNA, or a combination
of one or more of these can also be present in the cell. See also,
the section herein entitled "Orthogonal aminoacyl-tRNA
synthetases."
[0136] Methods of producing an orthogonal tRNA (O-tRNA) are known.
In certain embodiments of the invention, the O-tRNAs can be
produced by generating a library of mutants. The library of mutant
tRNAs can be generated using various mutagenesis techniques known
in the art. For example, the mutant tRNAs can be generated by
site-specific mutations, random point mutations, homologous
recombination, DNA shuffling or other recursive mutagenesis
methods, chimeric construction or any combination thereof, e.g., of
the example O-tRNA of SEQ ID NO: 1.
[0137] Additional mutations can be introduced at a specific
position(s), e.g., at a nonconservative position(s), or at a
conservative position, at a randomized position(s), or a
combination of both in a desired loop or region of a tRNA, e.g., an
anticodon loop, the acceptor stem, D arm or loop, variable loop,
T.PHI.C arm or loop, other regions of the tRNA molecule, or a
combination thereof. Typically, mutations in a tRNA include
mutating the anticodon loop of each member of the library of mutant
tRNAs to allow recognition of a selector codon. The method can
further include adding additional sequences to the O-tRNA.
Typically, an O-tRNA possesses an improvement of orthogonality for
a desired organism compared to the starting material, e.g., the
plurality of tRNA sequences, while preserving its affinity towards
a desired RS.
[0138] The methods optionally include analyzing the similarity
(and/or inferred homology) of sequences of tRNAs and/or
aminoacyl-tRNA synthetases to determine potential candidates for an
O-tRNA, O-RS and/or pairs thereof, that appear to be orthogonal for
a specific organism. Computer programs known in the art and
described herein can be used for the analysis, e.g., BLAST and
pileup programs can be used. In one example, to choose potential
orthogonal translational components for use in E. coli, a
synthetase and/or a tRNA is chosen that does not display close
sequence similarity to eubacterial organisms.
[0139] Typically, an O-tRNA is obtained by subjecting to, e.g.,
negative selection, a population of cells of a first species, where
the cells comprise a member of the plurality of potential O-tRNAs.
The negative selection eliminates cells that comprise a member of
the library of potential O-tRNAs that is aminoacylated by an
aminoacyl-tRNA synthetase (RS) that is endogenous to the cell. This
provides a pool of tRNAs that are orthogonal to the cell of the
first species.
[0140] In certain embodiments, in the negative selection, a
selector codon(s) is introduced into a polynucleotide that encodes
a negative selection marker, e.g., an enzyme that confers
antibiotic resistance, e.g., .beta.-lactamase, an enzyme that
confers a detectable product, e.g., .beta.-galactosidase,
chloramphenicol acetyltransferase (CAT), e.g., a toxic product,
such as barnase, at a nonessential position (e.g., still producing
a functional barnase), etc. Screening/selection is optionally done
by growing the population of cells in the presence of a selective
agent (e.g., an antibiotic, such as ampicillin). In one embodiment,
the concentration of the selection agent is varied.
[0141] For example, to measure the activity of suppressor tRNAs, a
selection system is used that is based on the in vivo suppression
of selector codon, e.g., nonsense (e.g., stop) or frameshift
mutations introduced into a polynucleotide that encodes a negative
selection marker, e.g., a gene for .beta.-lactamase (bla). For
example, polynucleotide variants, e.g., bla variants, with a
selector codon at a certain position (e.g., A184), are constructed.
Cells, e.g., bacteria, are transformed with these polynucleotides.
In the case of an orthogonal tRNA, which cannot be efficiently
charged by endogenous E. coli synthetases, antibiotic resistance,
e.g., ampicillin resistance, should be about or less than that for
a bacteria transformed with no plasmid. If the tRNA is not
orthogonal, or if a heterologous synthetase capable of charging the
tRNA is co-expressed in the system, a higher level of antibiotic,
e.g., ampicillin, resistance is be observed. Cells, e.g., bacteria,
are chosen that are unable to grow on LB agar plates with
antibiotic concentrations about equal to cells transformed with no
plasmids.
[0142] In the case of a toxic product (e.g., ribonuclease or
barnase), when a member of the plurality of potential tRNAs is
aminoacylated by endogenous host, e.g., Escherichia coli
synthetases (i.e., it is not orthogonal to the host, e.g.,
Escherichia coli synthetases), the selector codon is suppressed and
the toxic polynucleotide product produced leads to cell death.
Cells harboring orthogonal tRNAs or non-functional tRNAs
survive.
[0143] In one embodiment, the pool of tRNAs that are orthogonal to
a desired organism are then subjected to a positive selection in
which a selector codon is placed in a positive selection marker,
e.g., encoded by a drug resistance gene, such a .beta.-lactamase
gene. The positive selection is performed on a cell comprising a
polynucleotide encoding or comprising a member of the pool of tRNAs
that are orthogonal to the cell, a polynucleotide encoding a
positive selection marker, and a polynucleotide encoding a cognate
RS. In certain embodiments, the second population of cells
comprises cells that were not eliminated by the negative selection.
The polynucleotides are expressed in the cell and the cell is grown
in the presence of a selection agent, e.g., ampicillin. tRNAs are
then selected for their ability to be aminoacylated by the
coexpressed cognate synthetase and to insert an amino acid in
response to this selector codon. Typically, these cells show an
enhancement in suppression efficiency compared to cells harboring
non-functional tRNA(s), or tRNAs that cannot efficiently be
recognized by the synthetase of interest. The cell harboring the
non-functional tRNAs or tRNAs that are not efficiently recognized
by the synthetase of interest, are sensitive to the antibiotic.
Therefore, tRNAs that: (i) are not substrates for endogenous host,
e.g., Escherichia coli, synthetases; (ii) can be aminoacylated by
the synthetase of interest; and (iii) are functional in
translation, survive both selections.
[0144] Accordingly, the same marker can be either a positive or
negative marker, depending on the context in which it is screened.
That is, the marker is a positive marker if it is screened for, but
a negative marker if screened against.
[0145] The stringency of the selection, e.g., the positive
selection, the negative selection or both the positive and negative
selection, in the above described-methods, optionally includes
varying the selection stringency. For example, because barnase is
an extremely toxic protein, the stringency of the negative
selection can be controlled by introducing different numbers of
selector codons into the barnase gene and/or by using an inducible
promoter. In another example, the concentration of the selection or
screening agent is varied (e.g., ampicillin concentration). In some
aspects of the invention, the stringency is varied because the
desired activity can be low during early rounds. Thus, less
stringent selection criteria are applied in early rounds and more
stringent criteria are applied in later rounds of selection. In
certain embodiments, the negative selection, the positive selection
or both the negative and positive selection can be repeated
multiple times. Multiple different negative selection markers,
positive selection markers or both negative and positive selection
markers can be used. In certain embodiments, the positive and
negative selection marker can be the same.
[0146] Other types of selections/screening can be used in the
invention for producing orthogonal translational components, e.g.,
an O-tRNA, an O-RS, and an O-tRNA/O-RS pair that loads an unnatural
amino acid in response to a selector codon. For example, the
negative selection marker, the positive selection marker or both
the positive and negative selection markers can include a marker
that fluoresces or catalyzes a luminescent reaction in the presence
of a suitable reactant. In another embodiment, a product of the
marker is detected by fluorescence-activated cell sorting (FACS) or
by luminescence. Optionally, the marker includes an affinity based
screening marker. See also, Francisco et al., (1993) "Production
and fluorescence-activated cell sorting of Escherichia coli
expressing a functional antibody fragment on the external surface,"
Proc Natl Acad Sci U S A. 90:10444-8.
[0147] Additional methods for producing a recombinant orthogonal
tRNA can be found, e.g., in International Application Publications
WO 2002/086075, entitled "METHODS AND COMPOSITIONS FOR THE
PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS;" WO
2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE;" and
WO 2005/019415, filed Jul. 7, 2004. See also Forster et al., (2003)
"Programming peptidomimetic synthetases by translating genetic
codes designed de novo," PNAS 100(11):6353-6357; and, Feng et al.,
(2003), "Expanding tRNA recognition of a tRNA synthetase by a
single amino acid change," PNAS 100(10): 5676-5681.
Orthogonal aminoacyl-tRNA synthetase (O-RS)
[0148] An O-RS finding use with the invention preferentially
aminoacylates an O-tRNA with an unnatural amino acid, in vitro or
in vivo. An O-RS can be provided to the translation system, e.g., a
cell, by a polypeptide that includes an O-RS and/or by a
polynucleotide that encodes an O-RS or a portion thereof. For
example, an O-RS comprises an amino acid sequence as set forth in
the sequence listing and examples herein (see, e.g., FIG. 2, and
SEQ ID NO: 4-7), or a conservative variation thereof. In another
example, an O-RS, or a portion thereof, is encoded by a
polynucleotide sequence that encodes an amino acid comprising
sequence in the sequence listing or examples herein, or a
complementary polynucleotide sequence thereof. See, e.g., the
tables and examples herein for sequences of useful O-RS molecules.
See also, the section entitled "Nucleic Acid and Polypeptide
Sequence and Variants" herein.
[0149] Methods for identifying an orthogonal aminoacyl-tRNA
synthetase (O-RS), e.g., an O-RS, for use with an O-tRNA, are
known. For example, a method includes subjecting to selection,
e.g., positive selection, a population of cells of a first species,
where the cells individually comprise: 1) a member of a plurality
of aminoacyl-tRNA synthetases (RSs), (e.g., the plurality of RSs
can include mutant RSs, RSs derived from a species other than the
first species or both mutant RSs and RSs derived from a species
other than the first species); 2) the orthogonal tRNA (O-tRNA)
(e.g., from one or more species); and 3) a polynucleotide that
encodes an (e.g., positive) selection marker and comprises at least
one selector codon. Cells are selected or screened for those that
show an enhancement in suppression efficiency compared to cells
lacking or with a reduced amount of the member of the plurality of
RSs. Suppression efficiency can be measured by techniques known in
the art and as described herein. Cells having an enhancement in
suppression efficiency comprise an active RS that aminoacylates the
O-tRNA. A level of aminoacylation (in vitro or in vivo) by the
active RS of a first set of tRNAs from the first species is
compared to the level of aminoacylation (in vitro or in vivo) by
the active RS of a second set of tRNAs from the second species. The
level of aminoacylation can be determined by a detectable substance
(e.g., a labeled unnatural amino acid). The active RS that more
efficiently aminoacylates the second set of tRNAs compared to the
first set of tRNAs is typically selected, thereby providing an
efficient (optimized) orthogonal aminoacyl-tRNA synthetase for use
with the O-tRNA. An O-RS, identified by the method, is also a
feature of the invention.
[0150] Any of a number of assays can be used to determine
aminoacylation. These assays can be performed in vitro or in vivo.
For example, in vitro aminoacylation assays are described in, e.g.,
Hoben and Soll (1985) Methods Enzymol. 113:55-59. Aminoacylation
can also be determined by using a reporter along with orthogonal
translation components and detecting the reporter in a cell
expressing a polynucleotide comprising at least one selector codon
that encodes a protein. See also, WO 2002/085923, entitled "IN VIVO
INCORPORATION OF UNNATURAL AMINO ACIDS;" and WO 2004/094593,
entitiled "EXPANDING THE EUKARYOTIC GENETIC CODE."
[0151] Identified O-RS can be further manipulated to alter
substrate specificity of the synthetase, so that only a desired
unnatural amino acid, but not any of the common 20 amino acids, are
charged to the O-tRNA. Methods to generate an orthogonal aminoacyl
tRNA synthetase with a substrate specificity for an unnatural amino
acid include mutating the synthetase, e.g., at the active site in
the synthetase, at the editing mechanism site in the synthetase, at
different sites by combining different domains of synthetases, or
the like, and applying a selection process. A strategy is used,
which is based on the combination of a positive selection followed
by a negative selection. In the positive selection, suppression of
the selector codon introduced at a nonessential position(s) of a
positive marker allows cells to survive under positive selection
pressure. In the presence of both natural and unnatural amino
acids, survivors thus encode active synthetases charging the
orthogonal suppressor tRNA with either a natural or unnatural amino
acid. In the negative selection, suppression of a selector codon
introduced at a nonessential position(s) of a negative marker
removes synthetases with natural amino acid specificities.
Survivors of the negative and positive selection encode synthetases
that aminoacylate (charge) the orthogonal suppressor tRNA with
unnatural amino acids only. These synthetases can then be subjected
to further mutagenesis, e.g., DNA shuffling or other recursive
mutagenesis methods.
[0152] A library of mutant O-RSs can be generated using various
mutagenesis techniques known in the art. For example, the mutant
RSs can be generated by site-specific mutations, random point
mutations, homologous recombination, DNA shuffling or other
recursive mutagenesis methods, chimeric construction or any
combination thereof. For example, a library of mutant RSs can be
produced from two or more other, e.g., smaller, less diverse
"sub-libraries." Chimeric libraries of RSs are also included in the
invention. It should be noted that libraries of tRNA synthetases
from various organism (e.g., microorganisms such as eubacteria or
archaebacteria) such as libraries that comprise natural diversity
(see, e.g., U.S. Pat. No. 6,238,884 to Short et al. U.S. Pat. No.
5,756,316 to Schallenberger et al. U.S. Pat. No. 5,783,431 to
Petersen et al. U.S. Pat. No. 5,824,485 to Thompson et al. U.S.
Pat. No. 5,958,672 to Short et al), are optionally constructed and
screened for orthogonal pairs.
[0153] Once the synthetases are subject to the positive and
negative selection/screening strategy, these synthetases can then
be subjected to further mutagenesis. For example, a nucleic acid
that encodes the O-RS can be isolated; a set of polynucleotides
that encode mutated O-RSs (e.g., by random mutagenesis,
site-specific mutagenesis, recombination or any combination
thereof) can be generated from the nucleic acid; and, these
individual steps or a combination of these steps can be repeated
until a mutated O-RS is obtained that preferentially aminoacylates
the O-tRNA with the unnatural amino acid. In some aspects of the
invention, the steps are performed multiple times, e.g., at least
two times.
[0154] Additional levels of selection/screening stringency can also
be used in the methods of the invention, for producing O-tRNA,
O-RS, or pairs thereof. The selection or screening stringency can
be varied on one or both steps of the method to produce an O-RS.
This could include, e.g., varying the amount of selection/screening
agent that is used, etc. Additional rounds of positive and/or
negative selections can also be performed. Selecting or screening
can also comprise one or more of a change in amino acid
permeability, a change in translation efficiency, a change in
translational fidelity, etc. Typically, the one or more change is
based upon a mutation in one or more gene in an organism in which
an orthogonal tRNA-tRNA synthetase pair is used to produce
protein.
[0155] Additional general details for producing O-RS, and altering
the substrate specificity of the synthetase can be found in
Internal Publication Number WO 2002/086075, entitled "METHODS AND
COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA
SYNTHETASE PAIRS;" and WO 2004/094593, entitled "EXPANDING THE
EUKARYOTIC GENETIC CODE." See also, Wang and Schultz "Expanding the
Genetic Code," Angewandte Chemie Int. Ed., 44(1):34-66 (2005), the
content of which is incorporated by reference in its entirety.
Source and Hose Organisms
[0156] The orthogonal translational components (O-tRNA and O-RS)
finding use with the invention can be derived from any organism (or
a combination of organisms) for use in a host translation system
from any other species, with the caveat that the O-tRNA/O-RS
components and the host system work in an orthogonal manner. It is
not a requirement that the O-tRNA and the O-RS from an orthogonal
pair be derived from the same organism. In some aspects, the
orthogonal components are derived from Archaea genes (i.e.,
archaebacteria) for use in a eubacterial host system.
[0157] For example, the orthogonal O-tRNA can be derived from an
Archae organism, e.g., an archaebacterium, such as Methanococcus
jannaschii, Methanobacterium thennoautotrophicum, Halobacterium
such as Haloferax volcanii and Halobacterium species NRC-1,
Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii,
Aeuropyrum pernix, Methanococcus maripaludis, Methanopyrus
kandleri, Methanosarcina mazei (Mm), Pyrobaculum aerophilum,
Pyrococcus abyssi, Sulfolobus solfataricus (Ss), Sulfolobus
tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium, or the
like, or a eubacterium, such as Escherichia coli, Thermus
thermophilus, Bacillus stearothermphilus, or the like, while the
orthogonal O-RS can be derived from an organism or combination of
organisms, e.g., an archaebacterium, such as Methanococcus
jannaschii, Methanobacterium thermoautotrophicum, Halobacterium
such as Haloferax volcanii and Halobacterium species NRC-1,
Archaeoglobus fulgidus, Pyrococcus uriosus, Pyrococcus horikoshii,
Aeuropyrum pernix, Methanococcus maripaludis, Methanopyrus
kandleri, Methanosarcina mazei, Pyrobaculum aerophilum, Pyrococcus
abyssi, Sulfolobus solfataricus, Sulfolobus tokodaii, Thermoplasma
acidophilum, Thermoplasma volcanium, or the like, or a eubacterium,
such as Escherichia coli, Thermus thermophilus, Bacillus
stearothermphilus, or the like. In one embodiment, eukaryotic
sources, e.g., plants, algae, protists, fungi, yeasts, animals
(e.g., mammals, insects, arthropods, etc.), or the like, can also
be used as sources of O-tRNAs and O-RSs.
[0158] The individual components of an O-tRNA/O-RS pair can be
derived from the same organism or different organisms. In one
embodiment, the O-tRNA/O-RS pair is from the same organism.
Alternatively, the O-tRNA and the O-RS of the O-tRNA/O-RS pair are
from different organisms.
[0159] The O-tRNA, O-RS or O-tRNA/O-RS pair can be selected or
screened in vivo or in vitro and/or used in a cell, e.g., a
eubacterial cell, to produce a polypeptide with an unnatural amino
acid. The eubacterial cell used is not limited, for example,
Escherichia coli, Thermus thermophilus, Bacillus stearothermphilus,
or the like. Compositions of eubacterial cells comprising
translational components of the invention are also a feature of the
invention.
[0160] See also, International Application Publication Number WO
2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE,"
filed Apr. 16,2004, for screening O-tRNA and/or O-RS in one species
for use in another species.
[0161] In some aspects, the O-tRNA, O-RS or O-tRNA/O-RS pair can be
selected or screened in vivo or in vitro and/or used in a cell,
e.g., a eukaryotic cell, to produce a polypeptide with an unnatural
amino acid. The eukaryotic cell used is not limited; for example,
any suitable yeast cell, such as Saccharomyces cerevisiae (S.
cerevisiae) or the like, can be used. Compositions of eukaryotic
cells comprising translational components of the invention are also
a feature of the invention.
[0162] Although orthogonal translation systems (e.g., comprising an
O-RS, an O-tRNA and an unnatural amino acid) can utilize cultured
host cells to produce proteins having unnatural amino acids, it is
not intended that an orthogonal translation system of the invention
require an intact, viable host cell. For example, an orthogonal
translation system can utilize a cell-free system in the presence
of a cell extract. Indeed, the use of cell free, in vitro
transcription/translation systems for protein production is a well
established technique. Adaptation of these in vitro systems to
produce proteins having unnatural amino acids using orthogonal
translation system components described herein is well within the
scope of the invention.
Selector Codons
[0163] Selector codons in orthogonal translation systems expand the
genetic codon framework of protein biosynthetic machinery. For
example, a selector codon includes, e.g., a unique three base
codon, a nonsense codon, such as a stop codon, e.g., an amber codon
(UAG), or an opal codon (UGA), an unnatural codon, at least a four
base codon, a rare codon, or the like. A number of selector codons
can be introduced into a desired gene, e.g., one or more, two or
more, more than three, etc. By using different selector codons,
multiple orthogonal tRNA/synthetase pairs can be used that allow
the simultaneous site-specific incorporation of multiple unnatural
amino acids e.g., including at least one unnatural amino acid,
using these different selector codons.
[0164] In one embodiment, the methods involve the use of a selector
codon that is a stop codon for the incorporation of an unnatural
amino acid in vivo in a cell into a phage-displayed polypeptide
that is the target of post-translational modification. For example,
an O-tRNA is produced that recognizes the stop codon and is
aminoacylated by an O-RS with an unnatural amino acid. This O-tRNA
is not recognized by the naturally occurring host's aminoacyl-tRNA
synthetases. Conventional site-directed mutagenesis can be used to
introduce the stop codon at the site of interest in a
polynucleotide encoding a polypeptide of interest. See, e.g.,
Sayers, J. R., et al., (1988), "5',3' Exonuclease in
phosphorothioate-based oligonucleotide-directed mutagenesis,"
Nucleic Acids Res, 791-802. When the O-RS, O-tRNA and the nucleic
acid that encodes a polypeptide of interest are combined, e.g., in
vivo, the unnatural amino acid is incorporated in response to the
stop codon to give a polypeptide containing the unnatural amino
acid at the specified position. In one embodiment of the invention,
the stop codon used as a selector codon is an amber codon, UAG,
and/or an opal codon, UGA. In one example, a genetic code in which
UAG and UGA are both used as a selector codon can encode 22 amino
acids while preserving the ochre nonsense codon, UAA, which is the
most abundant termination signal.
[0165] The incorporation of unnatural amino acids in vivo can be
done without significant perturbation of the host cell. For example
in non-eukaryotic cells, such as Escherichia coli, because the
suppression efficiency for the UAG codon depends upon the
competition between the O-tRNA, e.g., the amber suppressor tRNA,
and the release factor 1 (RF1) (which binds to the UAG codon and
initiates release of the growing peptide from the ribosome), the
suppression efficiency can be modulated by, e.g., either increasing
the expression level of O-tRNA, e.g., the suppressor tRNA, or using
an RF1 deficient strain. In eukaryotic cells, because the
suppression efficiency for the UAG codon depends upon the
competition between the O-tRNA, e.g., the amber suppressor tRNA,
and a eukaryotic release factor (e.g., eRF) (which binds to a stop
codon and initiates release of the growing peptide from the
ribosome), the suppression efficiency can be modulated by, e.g.,
increasing the expression level of O-tRNA, e.g., the suppressor
tRNA. In addition, additional compounds can also be present, e.g.,
reducing agents such as dithiothretiol (DTT).
[0166] Unnatural amino acids can also be encoded with rare codons.
For example, when the arginine concentration in an in vitro protein
synthesis reaction is reduced, the rare arginine codon, AGG, has
proven to be efficient for insertion of Ala by a synthetic tRNA
acylated with alanine. See, e.g., Ma et al., Biochemistry, 32:7939
(1993). In this case, the synthetic tRNA competes with the
naturally occurring tRNA.sup.Arg, which exists as a minor species
in Escherichia coli. In addition, some organisms do not use all
triplet codons. An unassigned codon AGA in Micrococcus luteus has
been utilized for insertion of amino acids in an in vitro
transcription/translation extract. See, e.g., Kowal and Oliver,
Nucl. Acid. Res., 25:4685 (1997). Components of the invention can
be generated to use these rare codons in vivo.
[0167] Selector codons can also comprise extended codons, e.g.,
four or more base codons, such as, four, five, six or more base
codons. Examples of four base codons include, e.g., AGGA, CUAG,
UAGA, CCCU, and the like. Examples of five base codons include,
e.g., AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like.
Methods of the invention include using extended codons based on
frameshift suppression. Four or more base codons can insert, e.g.,
one or multiple unnatural amino acids, into the same protein. In
other embodiments, the anticodon loops can decode, e.g., at least a
four-base codon, at least a five-base codon, or at least a six-base
codon or more. Since there are 256 possible four-base codons,
multiple unnatural amino acids can be encoded in the same cell
using a four or more base codon. See also, Anderson et al., (2002)
"Exploring the Limits of Codon and Anticodon Size," Chemistry and
Biology, 9:237-244; and, Magliery (2001) "Expanding the Genetic
Code: Selection of Efficient Suppressors of Four-base Codons and
Identification of "Shifty" Four-base Codons with a Library Approach
in Escherichia coli, " J. Mol. Biol. 307: 755-769.
[0168] For example, four-base codons have been used to incorporate
unnatural amino acids into proteins using in vitro biosynthetic
methods. See, e.g., Ma et al., (1993) Biochemistry, 32:7939; and
Hohsaka et al., (1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU
were used to simultaneously incorporate 2-naphthylalanine and an
NBD derivative of lysine into streptavidin in vitro with two
chemically acylated frameshift suppressor tRNAs. See, e.g., Hohsaka
et al., (1999) J. Am. Chem. Soc., 121:12194. In an in vivo study,
Moore et al. examined the ability of tRNA.sup.Leu derivatives with
NCUA anticodons to suppress UAGN codons (N can be U, A, G, or C),
and found that the quadruplet UAGA can be decoded by a tRNA.sup.Leu
with a UCUA anticodon with an efficiency of 13 to 26% with little
decoding in the 0 or -1 frame. See Moore et al., (2000) J. Mol.
Biol., 298:195. In one embodiment, extended codons based on rare
codons or nonsense codons can be used in invention, which can
reduce missense readthrough and frameshift suppression at other
unwanted sites. Four base codons have been used as selector codons
in a variety of orthogonal systems. See, e.g., WO 2005/019415; WO
2005/007870 and WO 2005/07624. See also, Wang and Schultz
"Expanding the Genetic Code," Angewandte Chemie Int. Ed.,
44(1):34-66 (2005), the content of which is incorporated by
reference in its entirety. While the examples below utilize an
amber selector codon, four or more base codons can be used as well,
by modifying the examples herein to include four-base O-tRNAs and
synthetases modified to include mutations similar to those
previously described for various unnatural amino acid O-RSs.
[0169] For a given system, a selector codon can also include one of
the natural three base codons, where the endogenous system does not
use (or rarely uses) the natural base codon. For example, this
includes a system that is lacking a tRNA that recognizes the
natural three base codon, and/or a system where the three base
codon is a rare codon.
[0170] Selector codons optionally include unnatural base pairs.
These unnatural base pairs further expand the existing genetic
alphabet. One extra base pair increases the number of triplet
codons from 64 to 125. Properties of third base pairs include
stable and selective base pairing, efficient enzymatic
incorporation into DNA with high fidelity by a polymerase, and the
efficient continued primer extension after synthesis of the nascent
unnatural base pair. Descriptions of unnatural base pairs which can
be adapted for methods and compositions include, e.g., Hirao, et
al., (2002) "An unnatural base pair for incorporating amino acid
analogues into protein," Nature Biotechnology, 20:177-182. See also
Wu, Y., et al., (2002) J. Am. Chem. Soc. 124:14626-14630. Other
relevant publications are listed below.
[0171] For in vivo usage, the unnatural nucleoside is membrane
permeable and is phosphorylated to form the corresponding
triphosphate. In addition, the increased genetic information is
stable and not destroyed by cellular enzymes. Previous efforts by
Benner and others took advantage of hydrogen bonding patterns that
are different from those in canonical Watson-Crick pairs, the most
noteworthy example of which is the iso-C:iso-G pair. See, e.g.,
Switzer et al., (1989) J. Am. Chem. Soc., 111:8322; and Piccirilli
et al., (1990) Nature, 343:33; Kool, (2000) Curr. Opin. Chem.
Biol., 4:602. These bases in general mispair to some degree with
natural bases and cannot be enzymatically replicated. Kool and
co-workers demonstrated that hydrophobic packing interactions
between bases can replace hydrogen bonding to drive the formation
of base pair. See Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and
Guckian and Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In
an effort to develop an unnatural base pair satisfying all the
above requirements, Schultz, Romesberg and co-workers have
systematically synthesized and studied a series of unnatural
hydrophobic bases. A PICS:PICS self-pair is found to be more stable
than natural base pairs, and can be efficiently incorporated into
DNA by Klenow fragment of Escherichia coli DNA polymerase I (KF).
See, e.g., McMinn et al., (1999) J. Am. Chem. Soc., 121:11586; and
Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274. A 3MN:3MN
self-pair can be synthesized by KF with efficiency and selectivity
sufficient for biological function. See, e.g., Ogawa et al., (2000)
J. Am. Chem. Soc., 122:8803. However, both bases act as a chain
terminator for further replication. A mutant DNA polymerase has
been recently evolved that can be used to replicate the PICS self
pair. In addition, a 7AI self pair can be replicated. See, e.g.,
Tae et al., (2001) J. Am. Chem. Soc., 123:7439. A novel metallobase
pair, Dipic:Py, has also been developed, which forms a stable pair
upon binding Cu(II). See Meggers et al., (2000) J. Am. Chem. Soc.,
122:10714. Because extended codons and unnatural codons are
intrinsically orthogonal to natural codons, the methods of the
invention can take advantage of this property to generate
orthogonal tRNAs for them.
[0172] A translational bypassing system can also be used to
incorporate an unnatural amino acid in a desired polypeptide. In a
translational bypassing system, a large sequence is inserted into a
gene but is not translated into protein. The sequence contains a
structure that serves as a cue to induce the ribosome to hop over
the sequence and resume translation downstream of the
insertion.
Unnatural Amino Acids
[0173] As used herein, an unnatural amino acid refers to any amino
acid, modified amino acid, or amino acid analogue other than
selenocysteine and/or pyrrolysine and the following twenty
genetically encoded alpha-amino acids: alanine, arginine,
asparagine, aspartic acid, cysteine, glutamine, glutamic acid,
glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine,
valine. The generic structure of an alpha-amino acid is illustrated
by Formula I: ##STR1##
[0174] An unnatural amino acid is typically any structure having
Formula I wherein the R group is any substituent other than one
used in the twenty natural amino acids. See e.g., Biochemistry by
L. Stryer, 3.sup.rd ed. 1988, Freeman and Company, New York, for
structures of the twenty natural amino acids. Note that, the
unnatural amino acids of the invention can be naturally occurring
compounds other than the twenty alpha-amino acids above.
[0175] Because the unnatural amino acids of the invention typically
differ from the natural amino acids in side chain, the unnatural
amino acids form amide bonds with other amino acids, e.g., natural
or unnatural, in the same manner in which they are formed in
naturally occurring proteins. However, the unnatural amino acids
have side chain groups that distinguish them from the natural amino
acids.
[0176] Of particular interest herein are unnatural amino acids
provided in FIG. 3 and FIG. 15. For example, these unnatural amino
acids include but are not limited to aryl-azide amino acids, e.g.,
para-azido-L-phenylalanine, and alkynyl-amino acids, e.g.,
para-propargyloxyphenylalanine. Both the L and D-enantiomers of
these unnatural amino acids find use with the invention
[0177] In addition to these aryl-azide and alkynyl unnatural amino
acids, other unnatural amino acids can be simultaneously
incorporated into a phage-displayed polypeptide of interest, e.g.,
using an appropriate second O-RS/O-tRNA pair in conjunction with an
orthogonal pair to incorporate the aryl-azide or alkynyl unnatural
amino acid. Many such additional unnatural amino acids and suitable
orthogonal pair systems are known. See the references cited
herein.
[0178] In other unnatural amino acids, for example, R in Formula I
optionally comprises an alkyl-, aryl-, acyl-, hydrazine, cyano-,
halo-, hydrazide, alkenyl, ether, borate, boronate, phospho,
phosphono, phosphine, enone, imine, ester, hydroxylamine, amine,
and the like, or any combination thereof. Other unnatural amino
acids of interest include, but are not limited to, amino acids
comprising a photoactivatable cross-linker, spin-labeled amino
acids, fluorescent amino acids, metal binding amino acids,
metal-containing amino acids, radioactive amino acids, amino acids
with novel functional groups, amino acids that covalently or
noncovalently interact with other molecules, photocaged and/or
photoisomerizable amino acids, biotin or biotin-analogue containing
amino acids, keto containing amino acids, glycosylated amino acids,
a saccharide moiety attached to the amino acid side chain, amino
acids comprising polyethylene glycol or polyether, heavy atom
substituted amino acids, chemically cleavable or photocleavable
amino acids, amino acids with an elongated side chain as compared
to natural amino acids (e.g., polyethers or long chain
hydrocarbons, e.g., greater than about 5, greater than about 10
carbons, etc.), carbon-linked sugar-containing amino acids, amino
thioacid containing amino acids, and amino acids containing one or
more toxic moiety.
[0179] In another aspect, the invention provides unnatural amino
acids having the general structure illustrated by Formula IV below:
##STR2##
[0180] An unnatural amino acid having this structure is typically
any structure where R.sub.1 is a substituent used in one of the
twenty natural amino acids (e.g., tyrosine or phenylalanine) and
R.sub.2 is a substituent. Thus, this type of unnatural amino acid
can be viewed as a natural amino acid derivative.
[0181] In addition to unnatural amino acids that contain novel side
chains such as those shown in FIGS. 3 and 15, unnatural amino acids
can also optionally comprise modified backbone structures, e.g., as
illustrated by the structures of Formula II and III: ##STR3##
wherein Z typically comprises OH, NH.sub.2, SH, NH--R', or S--R'; X
and Y, which can be the same or different, typically comprise S or
0, and R and R', which are optionally the same or different, are
typically selected from the same list of constituents for the R
group described above for the unnatural amino acids having Formula
I as well as hydrogen. For example, unnatural amino acids of the
invention optionally comprise substitutions in the amino or
carboxyl group as illustrated by Formulas II and III. Unnatural
amino acids of this type include, but are not limited to,
.alpha.-hydroxy acids, .alpha.-thioacids
.alpha.-aminothiocarboxylates, e.g., with side chains corresponding
to the common twenty natural amino acids or unnatural side chains.
In addition, substitutions at the .alpha.-carbon optionally include
L, D, or .alpha.-.alpha.-disubstituted amino acids such as
D-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and
the like. Other structural alternatives include cyclic amino acids,
such as proline analogues as well as 3,4,6,7,8, and 9 membered ring
proline analogues, .beta. and .gamma. amino acids such as
substituted .beta.-alanine and .gamma.-amino butyric acid.
[0182] In some aspects, the invention utilizes unnatural amino
acids in the L-configuration. However, it is not intended that the
invention be limited to the use of L-configuration unnatural amino
acids. It is contemplated that the D-enantiomers of these unnatural
amino acids also find use with the invention.
[0183] Tyrosine analogs include para-substituted tyrosines,
ortho-substituted tyrosines, and meta substituted tyrosines,
wherein the substituted tyrosine comprises an alkynyl group, acetyl
group, a benzoyl group, an amino group, a hydrazine, an
hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a
methyl group, a C.sub.6-C.sub.20 straight chain or branched
hydrocarbon, a saturated or unsaturated hydrocarbon, an O-methyl
group, a polyether group, a nitro group, or the like. In addition,
multiply substituted aryl rings are also contemplated. Glutamine
analogs of the invention include, but are not limited to,
.alpha.-hydroxy derivatives, .gamma.-substituted derivatives,
cyclic derivatives, and amide substituted glutamine derivatives.
Example phenylalanine analogs include, but are not limited to,
para-substituted phenylalanines, ortho-substituted phenyalanines,
and meta-substituted phenylalanines, wherein the substituent
comprises an alkynyl group, a hydroxy group, a methoxy group, a
methyl group, an allyl group, an aldehyde, a nitro, a thiol group,
or keto group, or the like. Specific examples of unnatural amino
acids include, but are not limited to,
p-ethylthiocarbonyl-L-phenylalanine,
p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine,
7-amino-coumarin amino acid, 7-hydroxy-coumarin amino acid,
nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine,
p-carboxymethyl-L-phenylalanine, p-cyano-L-phenylalanine,
m-cyano-L-phenylalanine, biphenylalanine, 3-amino-L-tyrosine,
bipyridyl alanine, p-(2-amino-1-hydroxyethyl)-L-phenylalanine,
p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine and
p-nitro-L-phenylalanine. Also, a p-propargyloxyphenylalanine, a
3,4-dihydroxy-L-phenyalanine (DHP), a 3, 4,
6-trihydroxy-L-phenylalanine, a 3,4,5-trihydroxy-L-phenylalanine,
4-nitro-phenylalanine, a p-acetyl-L-phenylalanine,
O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a
3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a
4-propyl-L-tyrosine, a 3-nitro-tyrosine, a 3-thiol-tyrosine, a
tri-O-acetyl-GlcNAc.beta.-serine, an L-Dopa, a fluorinated
phenylalanine, an isopropyl-L-phenylalanine, a
p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a
p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a
phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine,
a p-amino-L-phenylalanine, and an isopropyl-L-phenylalanine, and
the like. The structures of a variety of unnatural amino acids that
can be incorporated using orthogonal translation systems are known.
See the references cited herein, each of which is incorporated
herein by reference in its entirety.
Chemical Synthesis of Unnatural Amino Acids
[0184] Many of the unnatural amino acids provided above are
commercially available, e.g., from Sigma (USA) or Aldrich
(Milwaukee, Wis., USA). Those that are not commercially available
are optionally synthesized as provided in various publications or
using standard methods known to those of skill in the art. For
organic synthesis techniques, see, e.g., Organic Chemistry by
Fessendon and Fessendon, (1982, Second Edition, Willard Grant
Press, Boston Mass.); Advanced Organic Chemistry by March (Third
Edition, 1985, Wiley and Sons, New York); and Advanced Organic
Chemistry by Carey and Sundberg (Third Edition, Parts A and B,
1990, Plenum Press, New York). Additional publications describing
the synthesis of unnatural amino acids include, e.g., WO
2002/085923 entitled "In vivo incorporation of Unnatural Amino
Acids;" Matsoukas et al., (1995) J. Med. Chem., 38, 4660-4669; King
and Kidd, (1949) "A New Synthesis of Glutamine and of
.gamma.-Dipeptides of Glutamic Acid from Phthylated
Intermediates,". J. Chem. Soc. 3315-3319; Friedman, and Chatterrji
(1959) "Synthesis of Derivatives of Glutamine as Model Substrates
for Anti-Tumor Agents," J. Am. Chem. Soc. 81, 3750-3752; Craig et
al., (1988) "Absolute Configuration of the Enantiomers of
7-Chloro-4 [[4-(diethylamino)-1-methylbutyl]amino]quinoline
(Chloroquine)," J. Org. Chem. 53, 1167-1170; Azoulay et al. (1991)
"Glutamine analogues as Potential Antimalarials," Eur. J. Med.
Chem. 26, 201-5; Koskinen and Rapoport (1989) "Synthesis of
4-Substituted Prolines as Conformationally Constrained Amino Acid
Analogues,". J. Org. Chem. 54, 1859-1866; Christie and Rapoport
(1985) "Synthesis of Optically Pure Pipecolates from L-Asparagine.
Application to the Total Synthesis of (+)-Apovincamine through
Amino Acid Decarbonylation and Iminium Ion Cyclization,". J. Org.
Chem. 1989:1859-1866; Barton et al., (1987) "Synthesis of Novel
a-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis of
L- and D-a-Amino-Adipic Acids, L-a-aminopimelic Acid and
Appropriate Unsaturated Derivatives," Tetrahedron Lett.
43:4297-4308; and, Subasinghe et al., (1992) "Quisqualic acid
analogues: synthesis of beta-heterocyclic 2-aminopropanoic acid
derivatives and their activity at a novel quisqualate-sensitized
site," J. Med. Chem. 35:4602-7. See also, International Publication
WO 2004/058946, entitled "PROTEIN ARRAYS," filed on Dec. 22,
2003.
Cellular Uptake of Unnatural Amino Acids
[0185] Unnatural amino acid uptake by a cell is one issue that is
typically considered when designing and selecting unnatural amino
acids, e.g., for incorporation into a protein. For example, the
high charge density of .alpha.-amino acids suggests that these
compounds are unlikely to be cell permeable. Natural amino acids
are taken up into the cell via a collection of protein-based
transport systems often displaying varying degrees of amino acid
specificity. A rapid screen can be done which assesses which
unnatural amino acids, if any, are taken up by cells. See, e.g.,
the toxicity assays in, e.g., International Publication WO
2004/058946, entitled "PROTEIN ARRAYS," filed on Dec. 22, 2003; and
Liu and Schultz (1999) "Progress toward the evolution of an
organism with an expanded genetic code," PNAS 96:4780-4785.
Although uptake is easily analyzed with various assays, an
alternative to designing unnatural amino acids that are amenable to
cellular uptake pathways is to provide biosynthetic pathways to
create amino acids in vivo.
Biosynthesis of Unnatural Amino Acids
[0186] Many biosynthetic pathways already exist in cells for the
production of amino acids and other compounds. While a biosynthetic
method for a particular unnatural amino acid may not exist in
nature, e.g., in a cell, the invention provides such methods. For
example, biosynthetic pathways for unnatural amino acids are
optionally generated in host cell by adding new enzymes or
modifying existing host cell pathways. Additional new enzymes are
optionally naturally occurring enzymes or artificially evolved
enzymes. For example, the biosynthesis of p-aminophenylalanine (as
presented in an example in WO 2002/085923, supra) relies on the
addition of a combination of known enzymes from other organisms.
The genes for these enzymes can be introduced into a cell by
transforming the cell with a plasmid comprising the genes. The
genes, when expressed in the cell, provide an enzymatic pathway to
synthesize the desired compound. Examples of the types of enzymes
that are optionally added are provided in the examples below.
Additional enzymes sequences are found, e.g., in Genbank.
Artificially evolved enzymes are also optionally added into a cell
in the same manner. In this manner, the cellular machinery and
resources of a cell are manipulated to produce unnatural amino
acids.
[0187] Indeed, any of a variety of methods can be used for
producing novel enzymes for use in biosynthetic pathways, or for
evolution of existing pathways, for the production of unnatural
amino acids, in vitro or in vivo. Many available methods of
evolving enzymes and other biosynthetic pathway components can be
applied to the present invention to produce unnatural amino acids
(or, indeed, to evolve synthetases to have new substrate
specificities or other activities of interest). For example, DNA
shuffling is optionally used to develop novel enzymes and/or
pathways of such enzymes for the production of unnatural amino
acids (or production of new synthetases), in vitro or in vivo. See,
e.g., Stemmer (1994), "Rapid evolution of a protein in vitro by DNA
shuffling," Nature 370(4):389-391; and, Stemmer, (1994),"DNA
shuffling by random fragmentation and reassembly: In vitro
recombination for molecular evolution," Proc. Natl. Acad. Sci.
USA., 91:10747-10751. A related approach shuffles families of
related (e.g., homologous) genes to quickly evolve enzymes with
desired characteristics. An example of such "family gene shuffling"
methods is found in Crameri et al., (1998) "DNA shuffling of a
family of genes from diverse species accelerates directed
evolution" Nature, 391(6664): 288-291. New enzymes (whether
biosynthetic pathway components or synthetases) can also be
generated using a DNA recombination procedure known as "incremental
truncation for the creation of hybrid enzymes" ("ITCHY"), e.g., as
described in Ostermeier et al., (1999) "A combinatorial approach to
hybrid enzymes independent of DNA homology" Nature Biotech 17:1205.
This approach can also be used to generate a library of enzyme or
other pathway variants which can serve as substrates for one or
more in vitro or in vivo recombination methods. See, also,
Ostermeier et al. (1999) "Combinatorial Protein Engineering by
Incremental Truncation," Proc. Natl. Acad. Sci. USA, 96: 3562-67,
and Ostermeier et al. (1999), "Incremental Truncation as a Strategy
in the Engineering of Novel Biocatalysts," Biological and Medicinal
Chemistry, 7:2139-44. Another approach uses exponential ensemble
mutagenesis to produce libraries of enzyme or other pathway
variants that are, e.g., selected for an ability to catalyze a
biosynthetic reaction relevant to producing an unnatural amino acid
(or a new synthetase). In this approach, small groups of residues
in a sequence of interest are randomized in parallel to identify,
at each altered position, amino acids which lead to functional
proteins. Examples of such procedures, which can be adapted to the
present invention to produce new enzymes for the production of
unnatural amino acids (or new synthetases) are found in Delegrave
and Youvan (1993) Biotechnology Research 11:1548-1552. In yet
another approach, random or semi-random mutagenesis using doped or
degenerate oligonucleotides for enzyme and/or pathway component
engineering can be used, e.g., by using the general mutagenesis
methods of e.g., Arkin and Youvan (1992) "Optimizing nucleotide
mixtures to encode specific subsets of amino acids for semi-random
mutagenesis" Biotechnology 10:297-300; or Reidhaar-Olson et al.
(1991) "Random mutagenesis of protein sequences using
oligonucleotide cassettes" Methods Enzymol. 208:564-86. Yet another
approach, often termed a "non-stochastic" mutagenesis, which uses
polynucleotide reassembly and site-saturation mutagenesis can be
used to produce enzymes and/or pathway components, which can then
be screened for an ability to perform one or more synthetase or
biosynthetic pathway function (e.g., for the production of
unnatural amino acids in vivo). See, e.g., Short "NON-STOCHASTIC
GENERATION OF GENETIC VACCINES AND ENZYMES" WO 00/46344.
[0188] An alternative to such mutational methods involves
recombining entire genomes of organisms and selecting resulting
progeny for particular pathway functions (often referred to as
"whole genome shuffling"). This approach can be applied to the
present invention, e.g., by genomic recombination and selection of
an organism (e.g., an E. coli or other cell) for an ability to
produce an unnatural amino acid (or intermediate thereof). For
example, methods taught in the following publications can be
applied to pathway design for the evolution of existing and/or new
pathways in cells to produce unnatural amino acids in vivo: Patnaik
et al. (2002) "Genome shuffling of lactobacillus for improved acid
tolerance" Nature Biotechnology, 20(7): 707-712; and Zhang et al.
(2002) "Genome shuffling leads to rapid phenotypic improvement in
bacteria" Nature, Feb. 7, 415(6872): 644-646.
[0189] Other techniques for organism and metabolic pathway
engineering, e.g., for the production of desired compounds are also
available and can also be applied to the production of unnatural
amino acids. Examples of publications teaching useful pathway
engineering approaches include: Nakamura and White (2003)
"Metabolic engineering for the microbial production of 1,3
propanediol" Curr. Opin. Biotechnol. 14(5):454-9; Berry et al.
(2002) "Application of Metabolic Engineering to improve both the
production and use of Biotech Indigo" J. Industrial Microbiology
and Biotechnology 28:127-133; Banta et al. (2002) "Optimizing an
artificial metabolic pathway: Engineering the cofactor specificity
of Corynebacterium 2,5-diketo-D-gluconic acid reductase for use in
vitamin C biosynthesis" Biochemistry, 41(20), 6226-36; Selivonova
et al. (2001) "Rapid Evolution of Novel Traits in Microorganisms"
Applied and Environmental Microbiology, 67:3645, and many
others.
[0190] Regardless of the method used, typically, the unnatural
amino acid produced with an engineered biosynthetic pathway of the
invention is produced in a concentration sufficient for efficient
protein biosynthesis, e.g., a natural cellular amount, but not to
such a degree as to significantly affect the concentration of other
cellular amino acids or to exhaust cellular resources. Typical
concentrations produced in vivo in this manner are about 10 mM to
about 0.05 mM. Once a cell is engineered to produce enzymes desired
for a specific pathway and an unnatural amino acid is generated, in
vivo selections are optionally used to further optimize the
production of the unnatural amino acid for both ribosomal protein
synthesis and cell growth.
Orthogonal Components Finding Use with the Invention
[0191] The invention provides phage that display polypeptides
comprising unnatural amino acids, where the unnatural amino acid
(and consequently the phage) are post-translationally modified. The
invention also provides methods for producing the modified
phage.
[0192] The incorporation of the unnatural amino acid into the
phage-displayed protein is accomplished by adapting orthogonal
pairs for the genetic encoding of unnatural amino acids into
proteins in E. coli, where the orthogonal components do not
cross-react with endogenous E. coli components of the translational
machinery of the host cell, but recognize the desired unnatural
amino acid and incorporate it into proteins in response to the
selector codon (e.g., an amber nonsense codon, TAG). The orthogonal
components finding use with the invention include orthogonal
aminoacyl-tRNA synthetases derived from Methanococcus jannaschii
tyrosyl tRNA-synthetase, and the mutant tyrosyl tRNA.sub.CUA amber
suppressor, which function as an orthogonal pair in a eubacterial
host cell such as E. coli. In this system, the mutant
aminoacyl-tRNA synthetases aminoacylate the suppressor tRNA with
its respective unnatural amino acid and not with any of the common
twenty amino acids.
[0193] Methods of producing orthogonal components find use with the
invention, where these methods result in the incorporation of
unnatural amino acids, e.g., the unnatural amino acids provided in
FIG. 3 and FIG. 15, into a growing phage-displayed polypeptide
chain in response to a selector codon, e.g., an amber stop codon, a
nonsense codon, a four or more base codon, etc., e.g., in vivo. For
example, orthogonal-tRNAs (O-tRNAs), orthogonal aminoacyl-tRNA
synthetases (O-RSs) and pairs thereof find use with the invention
These pairs can be used to incorporate an unnatural amino acid into
growing polypeptide chains, where the polypeptide is incorporated
into a phage-display system, and is subsequently
post-translationally modified.
[0194] An orthogonal aminoacyl-tRNA synthetase (O-RS) finding use
with the invention includes any O-RS the preferentially
aminoacylates an O-tRNA with an amino acid that can be specifically
and selectively post-translationally modified in a phage-display
system. These amino acids include, but are not limited to,
aryl-azide amino acids, e.g., para-azido-L-phenylalanine, and
alkynyl-amino acids, e.g., para-propargyloxyphenylalanine. For
example, the invention provides phage with a displayed polypeptide
comprising at least one post-translationally modified unnatural
amino acid residue, where the amino acid residue can be selectively
modified. Such amino acids include but are not limited to amino
acids with keto-moieties, for example, para-acetyl-L-phenylalanine,
meta-acetyl-L-phenylalanine and
para-(3-oxobutanoyl)-L-phenylalanine (see, e.g., Wang et al., Proc.
Natl. Acad. Sci. U.S.A. 2003, 100:56-61 and Liu et al., (2003) JACS
125(7):1702-1703). Additional unnatural amino acids with reactive
chemistries can also be incorporated into phage using orthogonal
translation systems, where the unnatural amino acid is a selective
target for modification. These systems can incorporate, e.g.,
para-(2-amino-1-hydroxyethyl)-L-phenylalanine,
para-isopropylthiocarbonyl-L-phenylalanine and
para-ethylthiocarbonyl-L-phenylalanine (see International
Application No. PCT/US2005/039210 by Schultz et al., filed Oct. 27,
2005, entitled "ORTHOGONAL TRANSLATION COMPONENTS FOR THE IN VIVO
INCORPORATION OF UNNATURAL AMINO ACIDS").
[0195] For additional information regarding unnatural amino acids
that can be post-translationally modified, see, for example, the
unnatural amino acid orthogonal systems described in Chin et al.,
Science (2003) 301:964-967; Zhang et al., Proc. Natl. Acad. Sci.
U.S.A. 2004, 101:8882-8887; Anderson et al., Proc. Natl. Acad. Sci.
U.S.A. 2004, 101:7566-7571; Wang et al., (2001) Science
292:498-500; Chin et al., (2002) Journal of the American Chemical
Society 124:9026-9027; Chin and Schultz, (2002) ChemBioChem
11:1135-1137; Chin, et al., (2002) PNAS United States of America
99:11020-11024; Wang and Schultz, (2002) Chem. Comm., 1-10; Wang
and Schultz "Expanding the Genetic Code," Angewandte Chemie Int.
Ed., 44(1):34-66 (2005); Xie and Schultz, "An Expanding Genetic
Code," Methods 36:227-238 (2005); and Deiters et al, Bioorganic
& Medicinal Chemistry Letters 15:1521-1524 (2005), each of
which is incorporated by reference in its entirety.
[0196] See also the unnatural amino acid orthogonal systems
described in International Publications WO 2002/086075, entitled
"METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA
AMINOACYL-tRNA SYNTHETASE PAIRS;" WO 2002/085923, entitled "IN VIVO
INCORPORATION OF UNNATURAL AMINO ACIDS;" WO 2004/094593, entitled
"EXPANDING THE EUKARYOTIC GENETIC CODE;" WO 2005/019415, filed Jul.
7, 2004; WO2005/007870, filed Jul. 7, 2004; WO 2005/007624, filed
Jul. 7, 2004; International Publication No. WO2006/034332, filed on
Sep. 20, 2005; and International Application No. PCT/US2005/039210
by Schultz et al., filed Oct. 27, 2005, entitled "ORTHOGONAL
TRANSLATION COMPONENTS FOR THE IN VIVO INCORPORATION OF UNNATURAL
AMINO ACIDS."
[0197] In certain embodiments, the O-RS finding use with the
invention preferentially aminoacylates the O-tRNA over any
endogenous tRNA with an the particular unnatural amino acid, where
the O-RS has a bias for the O-tRNA, and where the ratio of O-tRNA
charged with an unnatural amino acid to the endogenous tRNA charged
with the same unnatural amino acid is greater than 1:1, and more
preferably where the O-RS charges the O-tRNA exclusively or nearly
exclusively.
[0198] The invention also makes use of orthogonal tRNAs (O-tRNA),
where the O-tRNA recognizes a selector codon. Typically, an O-tRNA
includes at least about, e.g., a 45%, a 50%, a 60%, a 75%, an 80%,
or a 90% or more suppression efficiency in the presence of a
cognate synthetase in response to a selector codon as compared to
the suppression efficiency of an O-tRNA comprising or encoded by a
polynucleotide sequence as set forth in the sequence listings
(e.g., SEQ ID NO: 1). In one embodiment, the suppression efficiency
of the O-RS and the O-tRNA together is, e.g., 5 fold, 10 fold, 15
fold, 20 fold, 25 fold or more greater than the suppression
efficiency of the O-tRNA in the absence of an O-RS. In some
aspects, the suppression efficiency of the O-RS and the O-tRNA
together is at least 45% of the suppression efficiency of an
orthogonal tyrosyl-tRNA synthetase pair derived from Methanococcus
jannaschii.
[0199] The invention makes use of cells (e.g., E. coli) comprising
a translation system and nucleotide sequences that program phage
production, where the translation system includes an
orthogonal-tRNA (O-tRNA), an orthogonal aminoacyl-tRNA synthetase
(O-RS), and, an unnatural amino acid that can be
post-translationally modified following its incorporation into the
phage-displayed polypeptide. Typically, the O-RS preferentially
aminoacylates the O-tRNA over any endogenous tRNA with the
unnatural amino acid, where the O-RS has a bias for the O-tRNA, and
where the ratio of O-tRNA charged with the unnatural amino acid to
the endogenous tRNA charged with the unnatural amino acid is
greater than 1:1, and more preferably where the O-RS charges the
O-tRNA exclusively or nearly exclusively. The O-tRNA recognizes the
first selector codon, and the O-RS preferentially aminoacylates the
O-tRNA with an unnatural amino acid.
[0200] Various polynucleotides also find use with the invention.
These polynucleotides include an artificial (e.g., man-made, and
not naturally occurring, e.g., recombinant) polynucleotide
comprising a nucleotide sequence encoding an O-RS. A polynucleotide
finding use with the invention can also includes a nucleic acid
that hybridizes to a polynucleotide described above, under highly
stringent conditions, over substantially the entire length of the
nucleic acid. Vectors comprising polynucleotides also find use with
the invention. For example, a vector can include a plasmid, a
cosmid, a phage, a virus, an expression vector, and/or the like.
Methods for producing components of an O-tRNA/O-RS pair are known
and find use with the invention. See the present disclosure and the
reference cited herein.
Nucleic Acid and Polypeptide Sequence and Variants
[0201] As described herein, polynucleotide sequences encoding,
e.g., O-tRNAs and O-RSs, find use with the invention, as do the
respective amino acid sequences encoded by the polynucleotides. The
disclosure provides and references examples of polynucleotide and
polypeptide sequences that find use with the invention. However, it
will be appreciated that use of the invention is not limited to
those sequences disclosed herein. One of skill will appreciate that
the invention also provides many related sequences with the
functions described herein, e.g., polynucleotides and polypeptides
encoding conservative variants of an O-RS disclosed herein.
[0202] A polynucleotide finding use with the invention also
includes an artificial polynucleotide that is, e.g., at least 75%,
at least 80%, at least 90%, at least 95%, at least 98% or more
identical to that of a naturally occurring tRNA, (but is other than
a naturally occurring tRNA). A polynucleotide finding use with the
invention also includes an artificial polynucleotide that is, e.g.,
at least 75%, at least 80%, at least 90%, at least 95%, at least
98% or more identical (but not 100% identical) to that of a
naturally occurring tRNA.
[0203] In certain embodiments, a vector finding use with the
invention (e.g., a plasmid, a cosmid, a phage, a virus, etc.)
comprises a polynucleotide that finds use with the invention. In
some embodiments, the vector is an expression vector. In other
embodiments, the expression vector includes a promoter operably
linked to one or more of the polynucleotides of the invention. In
other embodiments, a cell comprises a vector that includes a
polynucleotide finding use with the invention.
[0204] One of skill will appreciate that many variants of the
disclosed sequences also find use with the invention. For example,
conservative variations of the disclosed sequences that yield a
functionally identical sequence find use with the invention.
Variants of the nucleic acid polynucleotide sequences, wherein the
variants hybridize to at least one disclosed sequence, find use
with the invention.
Conservative Variations
[0205] Owing to the degeneracy of the genetic code, "silent
substitutions" (i.e., substitutions in a nucleic acid sequence
which do not result in an alteration in an encoded polypeptide) are
an implied feature of every nucleic acid sequence that encodes an
amino acid sequence. Similarly, "conservative amino acid
substitutions," where one or a limited number of amino acids in an
amino acid sequence are substituted with different amino acids with
highly similar properties, are also readily identified as being
highly similar to a disclosed construct. Such conservative
variations of each disclosed sequence are a feature of the present
invention.
[0206] "Conservative variations" of a particular nucleic acid
sequence refers to those nucleic acids which encode identical or
essentially identical amino acid sequences, or, where the nucleic
acid does not encode an amino acid sequence, to essentially
identical sequences. One of skill will recognize that individual
substitutions, deletions or additions which alter, add or delete a
single amino acid or a small percentage of amino acids (typically
less than 5%, more typically less than 4%, 2% or 1%) in an encoded
sequence are "conservatively modified variations" where the
alterations result in the deletion of an amino acid, addition of an
amino acid, or substitution of an amino acid with a chemically
similar amino acid. Thus, "conservative variations" of a listed
polypeptide sequence of the present invention include substitutions
of a small percentage, typically less than 5%, more typically less
than 2% or 1%, of the amino acids of the polypeptide sequence, with
an amino acid of the same conservative substitution group. Finally,
the addition of sequences which do not alter the encoded activity
of a nucleic acid molecule, such as the addition of a
non-functional sequence, is a conservative variation of the basic
nucleic acid.
[0207] Conservative substitution tables providing functionally
similar amino acids are well known in the art, where one amino acid
residue is substituted for another amino acid residue having
similar chemical properties (e.g., aromatic side chains or
positively charged side chains), and therefore does not
substantially change the functional properties of the polypeptide
molecule. The following sets forth example groups that contain
natural amino acids of like chemical properties, where
substitutions within a group is a "conservative substitution".
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions
Positively Negatively Nonpolar and/ Polar, Charged Charged or
Aliphatic Uncharged Aromatic Side Side Side Chains Side Chains Side
Chains Chains Chains Glycine Serine Phenylalanine Lysine Aspartate
Alanine Threonine Tyrosine Arginine Glutamate Valine Cysteine
Tryptophan Histidine Leucine Methionine Isoleucine Asparagine
Proline Glutamine
Nucleic Acid Hybridization
[0208] Comparative hybridization can be used to identify nucleic
acids that find use with the invention, including conservative
variations of nucleic acids provided herein, and this comparative
hybridization method is a preferred method of distinguishing
nucleic acids that find use with the invention. Target nucleic
acids which hybridize to nucleic acids provided or referenced
herein under high, ultra-high and ultra-ultra high stringency
conditions also find use with the invention. Examples of such
nucleic acids include those with one or a few silent or
conservative nucleic acid substitutions as compared to a given
nucleic acid sequence.
[0209] A test nucleic acid is said to specifically hybridize to a
probe nucleic acid when it hybridizes at least 50% as well to the
probe as to the perfectly matched complementary target, i.e., with
a signal to noise ratio at least half as high as hybridization of
the probe to the target under conditions in which the perfectly
matched probe binds to the perfectly matched complementary target
with a signal to noise ratio that is at least about
5.times.-10.times. as high as that observed for hybridization to
any of the unmatched target nucleic acids.
[0210] Nucleic acids "hybridize" when they associate, typically in
solution. Nucleic acids hybridize due to a variety of well
characterized physico-chemical forces, such as hydrogen bonding,
solvent exclusion, base stacking and the like. An extensive guide
to the hybridization of nucleic acids is found in Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2,
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays," (Elsevier, New York), as well as in
Current Protocols in Molecular Biology, Ausubel et al., eds.,
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (supplemented
through 2004) ("Ausubel"); Hames and Higgins (1995) Gene Probes 1
IRL Press at Oxford University Press, Oxford, England, (Hames and
Higgins 1) and Hames and Higgins (1995) Gene Probes 2 IRL Press at
Oxford University Press, Oxford, England (Hames and Higgins 2)
provide details on the synthesis, labeling, detection and
quantification of DNA and RNA, including oligonucleotides.
[0211] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids which have more than
100 complementary residues on a filter in a Southern or northern
blot is 50% formalin with 1 mg of heparin at 42.degree. C., with
the hybridization being carried out overnight. An example of
stringent wash conditions is a 0.2.times.SSC wash at 65.degree. C.
for 15 minutes (see, Sambrook, supra for a description of SSC
buffer). Often the high stringency wash is preceded by a low
stringency wash to remove background probe signal. An example low
stringency wash is 2.times.SSC at 40.degree. C. for 15 minutes. In
general, a signal to noise ratio of 5x (or higher) than that
observed for an unrelated probe in the particular hybridization
assay indicates detection of a specific hybridization.
[0212] "Stringent hybridization wash conditions" in the context of
nucleic acid hybridization experiments such as Southern and
northern hybridizations are sequence dependent, and are different
under different environmental parameters. An extensive guide to the
hybridization of nucleic acids is found in Tijssen (1993), supra.
and in Hames and Higgins, 1 and 2. Stringent hybridization and wash
conditions can easily be determined empirically for any test
nucleic acid. For example, in determining stringent hybridization
and wash conditions, the hybridization and wash conditions are
gradually increased (e.g., by increasing temperature, decreasing
salt concentration, increasing detergent concentration and/or
increasing the concentration of organic solvents such as formalin
in the hybridization or wash), until a selected set of criteria are
met. For example, in highly stringent hybridization and wash
conditions, the hybridization and wash conditions are gradually
increased until a probe binds to a perfectly matched complementary
target with a signal to noise ratio that is at least 5.times. as
high as that observed for hybridization of the probe to an
unmatched target.
[0213] "Very stringent" conditions are selected to be equal to the
thermal melting point (T.sub.m) for a particular probe. The T.sub.m
is the temperature (under defined ionic strength and pH) at which
50% of the test sequence hybridizes to a perfectly matched probe.
For the purposes of the present invention, generally, "highly
stringent" hybridization and wash conditions are selected to be
about 5.degree. C. lower than the T.sub.m for the specific sequence
at a defined ionic strength and pH.
[0214] "Ultra high-stringency" hybridization and wash conditions
are those in which the stringency of hybridization and wash
conditions are increased until the signal to noise ratio for
binding of the probe to the perfectly matched complementary target
nucleic acid is at least 10.times. as high as that observed for
hybridization to any of the unmatched target nucleic acids. A
target nucleic acid which hybridizes to a probe under such
conditions, with a signal to noise ratio of at least 1/2 that of
the perfectly matched complementary target nucleic acid is said to
bind to the probe under ultra-high stringency conditions.
[0215] Similarly, even higher levels of stringency can be
determined by gradually increasing the hybridization and/or wash
conditions of the relevant hybridization assay. For example, those
in which the stringency of hybridization and wash conditions are
increased until the signal to noise ratio for binding of the probe
to the perfectly matched complementary target nucleic acid is at
least 10.times., 20.times., 50.times., 100.times., or 500.times. or
more as high as that observed for hybridization to any of the
unmatched target nucleic acids. A target nucleic acid which
hybridizes to a probe under such conditions, with a signal to noise
ratio of at least 1/2 that of the perfectly matched complementary
target nucleic acid is said to bind to the probe under
ultra-ultra-high stringency conditions.
[0216] Nucleic acids which do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code.
Unique Subsequences
[0217] In some aspects, the invention utilizes a nucleic acid that
comprises a unique subsequence in a nucleic acid selected from the
sequences of O-tRNAs and O-RSs disclosed or referenced herein. The
unique subsequence is unique as compared to a nucleic acid
corresponding to any known O-tRNA or O-RS nucleic acid sequence.
Alignment can be performed using, e.g., BLAST set to default
parameters. Any unique subsequence is useful, e.g., as a probe to
identify the nucleic acids of the invention.
[0218] Similarly, the invention utilizes a polypeptide which
comprises a unique subsequence in a polypeptide selected from the
sequences of O-RSs disclosed or referenced herein. Here, the unique
subsequence is unique as compared to a polypeptide corresponding to
any of known polypeptide sequence.
[0219] The invention also provides for target nucleic acids which
hybridizes under stringent conditions to a unique coding
oligonucleotide which encodes a unique subsequence in a polypeptide
selected from the sequences of O-RSs wherein the unique subsequence
is unique as compared to a polypeptide corresponding to any of the
control polypeptides (e.g., parental sequences from which
synthetases of the invention were derived, e.g., by mutation).
Unique sequences are determined as noted above.
Sequence Comparison, Identity, and Homology
[0220] The terms "identical" or "percent identity," in the context
of two or more nucleic acid or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same, when compared and aligned for maximum correspondence, as
measured using one of the sequence comparison algorithms described
below (or other algorithms available to persons of skill) or by
visual inspection.
[0221] The phrase "substantially identical," in the context of two
nucleic acids or polypeptides (e.g., DNAs encoding an O-tRNA or
O-RS, or the amino acid sequence of an O-RS) refers to two or more
sequences or subsequences that have at least about 60%, about 80%,
about 90-95%, about 98%, about 99% or more nucleotide or amino acid
residue identity, when compared and aligned for maximum
correspondence, as measured using a sequence comparison algorithm
or by visual inspection. Such "substantially identical" sequences
are typically considered to be "homologous," without reference to
actual ancestry. Preferably, the "substantial identity" exists over
a region of the sequences that is at least about 50 residues in
length, more preferably over a region of at least about 100
residues, and most preferably, the sequences are substantially
identical over at least about 150 residues, or over the full length
of the two sequences to be compared.
[0222] Proteins and/or protein sequences are "homologous" when they
are derived, naturally or artificially, from a common ancestral
protein or protein sequence. Similarly, nucleic acids and/or
nucleic acid sequences are homologous when they are derived,
naturally or artificially, from a common ancestral nucleic acid or
nucleic acid sequence. For example, any naturally occurring nucleic
acid can be modified by any available mutagenesis method to include
one or more selector codon. When expressed, this mutagenized
nucleic acid encodes a polypeptide comprising one or more unnatural
amino acid. The mutation process can, of course, additionally alter
one or more standard codon, thereby changing one or more standard
amino acid in the resulting mutant protein as well. Homology is
generally inferred from sequence similarity between two or more
nucleic acids or proteins (or sequences thereof). The precise
percentage of similarity between sequences that is useful in
establishing homology varies with the nucleic acid and protein at
issue, but as little as 25% sequence similarity is routinely used
to establish homology. Higher levels of sequence similarity, e.g.,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be
used to establish homology. Methods for determining sequence
similarity percentages (e.g., BLASTP and BLASTN using default
parameters) are described herein and are generally available.
[0223] For sequence comparison and homology determination,
typically one sequence acts as a reference sequence to which test
sequences are compared. When using a sequence comparison algorithm,
test and reference sequences are input into a computer, subsequence
coordinates are designated, if necessary, and sequence algorithm
program parameters are designated. The sequence comparison
algorithm then calculates the percent sequence identity for the
test sequence(s) relative to the reference sequence, based on the
designated program parameters.
[0224] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by
the search for similarity method of Pearson and Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection (see generally Current Protocols in Molecular Biology,
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
supplemented through 2004).
[0225] One example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity is the BLAST
algorithm, which is described in Altschul et al., J. Mol. Biol.
215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information website. This algorithm involves first identifying high
scoring sequence pairs (HSPs) by identifying short words of length
W in the query sequence, which either match or satisfy some
positive-valued threshold score T when aligned with a word of the
same length in a database sequence. T is referred to as the
neighborhood word score threshold (Altschul et al., supra). These
initial neighborhood word hits act as seeds for initiating searches
to find longer HSPs containing them. The word hits are then
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >O) and N
(penalty score for mismatching residues; always <O). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison
of both strands. For amino acid sequences, the BLASTP program uses
as defaults a wordlength (W) of 3, an expectation (E) of 10, and
the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc.
Natl. Acad. Sci. USA 89:10915).
[0226] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin and Altschul,
Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
Mutagenesis and Other Molecular Biology Techniques
[0227] Polynucleotide and polypeptides of the invention and used in
the invention can be manipulated using molecular biological
techniques. General texts which describe molecular biological
techniques include Berger and Kimmel, Guide to Molecular Cloning
Techniques Methods in Enzymology volume 152 Academic Press, Inc.,
San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning--A
Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 2001 ("Sambrook") and Current
Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc., (supplemented through 2004)
("Ausubel"). These texts describe mutagenesis, the use of vectors,
promoters and many other relevant topics related to, e.g., the
generation of genes that include selector codons for production of
proteins that include unnatural amino acids, orthogonal tRNAs,
orthogonal synthetases, and pairs thereof.
[0228] Various types of mutagenesis can be used in conjunction with
the invention, e.g., to mutate tRNA molecules, to produce libraries
of tRNAs, to produce libraries of synthetases, to insert selector
codons that encode an unnatural amino acids in a protein or
polypeptide of interest. They include but are not limited to
site-directed, random point mutagenesis, homologous recombination,
DNA shuffling or other recursive mutagenesis methods, chimeric
construction, mutagenesis using uracil containing templates,
oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA
mutagenesis, mutagenesis using gapped duplex DNA or the like, or
any combination thereof. Additional suitable methods include point
mismatch repair, mutagenesis using repair-deficient host strains,
restriction-selection and restriction-purification, deletion
mutagenesis, mutagenesis by total gene synthesis, double-strand
break repair, and the like. Mutagenesis, e.g., involving chimeric
constructs, is also included in the present invention. In one
embodiment, mutagenesis can be guided by known information of the
naturally occurring molecule or altered or mutated naturally
occurring molecule, e.g., sequence, sequence comparisons, physical
properties, crystal structure or the like.
[0229] Host cells are genetically engineered (e.g., transformed,
transduced or transfected) with the polynucleotides of the
invention or constructs which include a polynucleotide, e.g., a
vector, which can be, for example, a cloning vector or an
expression vector. For example, the coding regions for the
orthogonal tRNA, the orthogonal tRNA synthetase, and the protein to
be derivatized are operably linked to gene expression control
elements that are functional in the desired host cell. Typical
vectors contain transcription and translation terminators,
transcription and translation initiation sequences, and promoters
useful for regulation of the expression of the particular target
nucleic acid. The vectors optionally comprise generic expression
cassettes containing at least one independent terminator sequence,
sequences permitting replication of the cassette in eukaryotes, or
prokaryotes, or both (e.g., shuttle vectors) and selection markers
for both prokaryotic and eukaryotic systems. Vectors are suitable
for replication and/or integration in prokaryotes, eukaryotes, or
preferably both. See Giliman and Smith, Gene 8:81 (1979); Roberts,
et al., Nature 328:731 (1987); Schneider, B., et al., Protein Expr.
Purif. 6435:10 (1995); Ausubel, Sambrook, Berger (all supra). The
vector can be, for example, in the form of a plasmid, a bacterium,
a virus, a naked polynucleotide, or a conjugated polynucleotide.
The vectors are introduced into cells and/or microorganisms by
standard methods including electroporation (From et al., Proc.
Natl. Acad. Sci. USA 82, 5824 (1985), infection by viral vectors,
high velocity ballistic penetration by small particles with the
nucleic acid either within the matrix of small beads or particles,
or on the surface (Klein et al., Nature 327, 70-73 (1987)), and/or
the like.
[0230] A highly efficient and versatile single plasmid system was
developed for site-specific incorporation of unnatural amino acids
into proteins in response to the amber stop codon (UAG) in E. coli.
In the new system, the pair of M. jannaschii suppressor
tRNAtyr(CUA) and tyrosyl-tRNA synthetase are encoded in a single
plasmid, which is compatible with most E. coli expression vectors.
Monocistronic tRNA operon under control of proK promoter and
terminator was constructed for optimal secondary structure and tRNA
processing. Introduction of a mutated form of glnS promoter for the
synthetase resulted in a significant increase in both suppression
efficiency and fidelity. Increases in suppression efficiency were
also obtained by multiple copies of tRNA gene as well as by a
specific mutation (D286R) on the synthetase (Kobayashi et al.,
"Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA
synthetases for genetic code expansion," Nat. Struct. Biol.,
10(6):425-432 [2003]). The generality of the optimized system was
also demonstrated by highly efficient and accurate incorporation of
several different unnatural amino acids, whose unique utilities in
studying protein function and structure were previously proven.
[0231] A catalogue of Bacteria and Bacteriophages useful for
cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of
Bacteria and Bacteriophae (1996) Gherna et al. (eds) published by
the ATCC. Additional basic procedures for sequencing, cloning and
other aspects of molecular biology and underlying theoretical
considerations are also found in Sambrook (supra), Ausubel (supra),
and in Watson et al. (1992) Recombinant DNA Second Edition
Scientific American Books, NY. In addition, essentially any nucleic
acid (and virtually any labeled nucleic acid, whether standard or
non-standard) can be custom or standard ordered from any of a
variety of commercial sources, such as the Midland Certified
Reagent Company (Midland, Tex.), The Great American Gene Company
(Ramona, Calif.), ExpressGen Inc. (Chicago, Ill.), Operon
Technologies Inc. (Alameda, Calif.) and many others.
[0232] The engineered host cells can be cultured in conventional
nutrient media modified as appropriate for such activities as, for
example, screening steps, activating promoters or selecting
transformants. These cells can optionally be cultured into
transgenic organisms. Other useful references, e.g. for cell
isolation and culture (e.g., for subsequent nucleic acid isolation)
include Freshney (1994) Culture of Animal Cells. a Manual of Basic
Technique, third edition, Wiley- Liss, New York and the references
cited therein; Payne et al. (1992) Plant Cell and Tissue Culture in
Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg
and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;
Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin
Heidelberg New York) and Atlas and Parks (eds) The Handbook of
Microbiological Media (1993) CRC Press, Boca Raton, Fla.
Proteins and Polypeptides of Interest
[0233] Methods of producing a phage with a displayed fusion protein
comprising an unnatural amino acid (an aryl-azide amino acid or an
alkynyl-amino acid) at a specified position that is
post-translationally modified are also a feature of the invention.
For example, a method can include growing, in an appropriate
medium, the cell with the phage construct (e.g., in an E. coli
cell), where the cell comprises a nucleic acid that comprises at
least one selector codon and encodes a protein (the capsid fusion
protein); and, providing the unnatural amino acid; where the cell
further comprises: an orthogonal-tRNA (O-tRNA) that functions in
the cell and recognizes the selector codon; and, an orthogonal
aminoacyl-tRNA synthetase (O-RS) that preferentially aminoacylates
the O-tRNA with the unnatural amino acid. The phage so produced in
the E. coli comprises a displayed fusion protein having an
unnatural amino acid at the position corresponding to the selector
codon. That phage is then reacted under conditions where the
unnatural amino acid undergoes covalent modification, thereby
producing a post-translationally modified phage.
[0234] In certain embodiments, the O-RS comprises a bias for the
aminoacylation of the cognate O-tRNA over any endogenous tRNA in an
expression system. The relative ratio between O-tRNA and endogenous
tRNA that is charged by the O-RS, when the O-tRNA and O-RS are
present at equal molar concentrations, is greater than 1:1,
preferably at least about 2:1, more preferably 5:1, still more
preferably 10:1, yet more preferably 20:1, still more preferably
50:1, yet more preferably 75:1, still more preferably 95:1, 98:1,
99:1, 100: 1, 500:1, 1,000:1, 5,000:1 or higher.
[0235] In some embodiments, the phage-displayed,
post-translationally modified fusion proteins can be cleaved using
a suitable protease and a protease recognition sequence that has
been incorporated into the phage-displayed fusion protein. This
cleavage can result in the release of the protein of interest, or a
portion thereof, from the phage capsid. In some embodiments, the
protein of interest comprises an amino acid sequence that is at
least 75% identical to that of a therapeutic protein, a diagnostic
protein, an industrial enzyme, or portion thereof.
[0236] The phage with a displayed fusion protein comprising an
unnatural amino acid (e.g., an aryl-azide amino acid or an
alkynyl-amino acid) at a specified position that is
post-translationally modified is a feature of the invention. The
phage is produced in a cell, e.g., an E. coli cell. The O-tRNA/O-RS
pairs also reside in the cell and utilize the host cell's
translation machinery, which results in the in vivo incorporation
of an unnatural amino acid into a fusion protein in response to a
selector codon and displayed on the phage. The ability of an
O-tRNA/O-RS system to function in a host cell to incorporate a wide
variety of unnatural amino acids that can be post-translationally
modified is known. See, e.g., Chin et al., Science (2003)
301:964-967; Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 2004,
101:8882-8887; Anderson et al., Proc. Natl. Acad. Sci. U.S.A. 2004,
101:7566-7571; Wang et al., (2001) Science 292:498-500; Chin et
al., (2002) Journal of the American Chemical Society 124:9026-9027;
Chin and Schultz, (2002) ChemBioChem 11:1135-1137; Chin, et al.,
(2002) PNAS United States of America 99:11020-11024; Wang and
Schultz, (2002) Chem. Comm., 1-10; Wang and Schultz "Expanding the
Genetic Code," Angewandte Chemie Int. Ed., 44(1):34-66 (2005); xie
and Schultz, "An Expanding Genetic Code," Methods 36:227-238
(2005); and Deiters et al, Bioorganic & Medicinal Chemistry
Letters 15:1521-1524 (2005), each of which is incorporated by
reference in its entirety.
[0237] See also the unnatural amino acid orthogonal systems
described in International Publications WO 2002/086075, entitled
"METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA
AMINOACYL-tRNA SYNTHETASE PAIRS;" WO 2002/085923, entitled "IN VIVO
INCORPORATION OF UNNATURAL AMINO ACIDS;" WO 2004/094593, entitled
"EXPANDING THE EUKARYOTIC GENETIC CODE;" WO 2005/019415, filed Jul.
7, 2004; WO2005/007870, filed Jul. 7, 2004; WO 2005/007624, filed
Jul. 7, 2004; International Publication No. WO2006/034332, filed on
Sep. 20, 2005; and International Application No. PCT/US2005/039210
by Schultz et al., filed Oct. 27, 2005, entitled "ORTHOGONAL
TRANSLATION COMPONENTS FOR THE IN VIVO INCORPORATION OF UNNATURAL
AMINO ACIDS," each of which is incorporated by reference in its
entirety.
[0238] The incorporation of an unnatural amino acid can be done to,
e.g., tailor changes in protein structure and/or function, e.g., to
change size, acidity, nucleophilicity, hydrogen bonding,
hydrophobicity, accessibility of protease target sites, target to a
moiety (e.g., for a protein array), incorporation of labels or
reactive groups, etc. Proteins that include an unnatural amino acid
can have enhanced or even entirely new catalytic or physical
properties. For example, the following properties are optionally
modified by inclusion of an unnatural amino acid into a protein:
toxicity, biodistribution, structural properties, spectroscopic
properties, chemical and/or photochemical properties, catalytic
ability, half-life (e.g., serum half-life), ability to react with
other molecules, e.g., covalently or noncovalently, and the like.
The compositions including proteins that include at least one
unnatural amino acid are useful for, e.g., novel therapeutics,
diagnostics, catalytic enzymes, industrial enzymes, binding
proteins (e.g., antibodies), and e.g., the study of protein
structure and function. See, e.g., Dougherty, (2000) "Unnatural
Amino Acids as Probes of Protein Structure and Function," Current
Opinion in Chemical Biology, 4:645-652. Proteins that comprise an
unnatural amino acid that can be selectively post-translationally
modified (e.g., by a [3+2] cycloaddition or a Staudinger
modification) can be engineered to contain any desired
functionality that can be coupled to the reaction partner. The
nature of the reaction partner is not limited in any way, except
only that it comprise a suitable reactive moiety that results in a
covalent attachment to the unnatural amino acid residue in the
phage-displayed polypeptide.
[0239] In some aspects, a composition includes at least one
phage-displayed protein with at least one, e.g., at least two, at
least three, at least four, at least five, at least six, at least
seven, at least eight, at least nine, or at least ten or more
unnatural amino acids. The unnatural amino acids can be the same or
different, e.g., there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or
more different sites in the protein that comprise 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10 or more different unnatural amino acids. In another
aspect, a composition includes a phage-displayed protein with at
least one, but fewer than all, of a particular amino acid present
in the protein is an unnatural amino acid. For a given protein with
more than one unnatural amino acids, the unnatural amino acids can
be identical or different (e.g., the protein can include two or
more different types of unnatural amino acids, or can include two
of the same unnatural amino acid). For a given protein with more
than two unnatural amino acids, the unnatural amino acids can be
the same, different or a combination of a multiple unnatural amino
acid of the same kind with at least one different unnatural amino
acid.
[0240] Essentially any phage-displayed protein (or portion thereof)
that includes an unnatural amino acid (and any corresponding coding
nucleic acid, e.g., which includes one or more selector codons) can
be produced using the compositions and methods herein. No attempt
is made to identify the hundreds of thousands of known proteins,
any of which can be modified to include one or more unnatural amino
acid, e.g., by tailoring any available mutation methods to include
one or more appropriate selector codon in a relevant translation
system. Common sequence repositories for known proteins include
GenBank EMBL, DDBJ and the NCBI. Other repositories can easily be
identified by searching the internet.
[0241] Typically, the proteins are, e.g., at least 60%, at least
70%, at least 75%, at least 80%, at least 90%, at least 95%, or at
least 99% or more identical to any available protein (e.g., a
therapeutic protein, a diagnostic protein, an industrial enzyme, or
portion thereof, and the like), and they comprise one or more
unnatural amino acid. Examples of therapeutic, diagnostic, and
other proteins that can be modified to comprise one or more
unnatural amino acid can be found, but not limited to, those in
International Publications WO 2004/094593, filed Apr. 16, 2004,
entitled "Expanding the Eukaryotic Genetic Code;" and, WO
2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO
ACIDS." Examples of therapeutic, diagnostic, and other proteins
that can be modified to comprise one or more unnatural amino acids
include, but are not limited to, e.g., Alpha-1 antitrypsin,
Angiostatin, Antihemolytic factor, antibodies (further details on
antibodies are found below), Apolipoprotein, Apoprotein, Atrial
natriuretic factor, Atrial natriuretic polypeptide, Atrial
peptides, C-X-C chemokines (e.g., T39765, NAP-2, ENA-78, Gro-a,
Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG), Calcitonin, CC
chemokines (e.g., Monocyte chemoattractant protein-1, Monocyte
chemoattractant protein-2, Monocyte chemoattractant protein-3,
Monocyte inflammatory protein-i alpha, Monocyte inflammatory
protein-1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065,
T64262), CD40 ligand, C-kit Ligand, Collagen, Colony stimulating
factor (CSF), Complement factor 5a, Complement inhibitor,
Complement receptor 1, cytokines, (e.g., epithelial Neutrophil
Activating Peptide-78, GRO.alpha./MGSA, GRO.beta., GRO.gamma.,
MIP-1.alpha., MIP-1.delta., MCP-1), Epidermal Growth Factor (EGF),
Erythropoietin ("EPO"), Exfoliating toxins A and B, Factor IX,
Factor VII, Factor VIII, Factor X, Fibroblast Growth Factor (FGF),
Fibrinogen, Fibronectin, G-CSF, GM-CSF, Glucocerebrosidase,
Gonadotropin, growth factors, Hedgehog proteins (e.g., Sonic,
Indian, Desert), Hemoglobin, Hepatocyte Growth Factor (HGF),
Hirudin, Human serum albumin, Insulin, Insulin-like Growth Factor
(IGF), interferons (e.g., IFN-.alpha., IFN-.beta., IFN-.gamma.),
interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-s, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-11, IL-12, etc.), Keratinocyte Growth Factor (KGF),
Lactoferrin, leukemia inhibitory factor, Luciferase, Neurturin,
Neutrophil inhibitory factor (NIF), oncostatin M, Osteogenic
protein, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones
(e.g., Human Growth Hormone), Pleiotropin, Protein A, Protein G,
Pyrogenic exotoxins A, B, and C, Relaxin, Renin, SCF, Soluble
complement receptor I, Soluble I-CAM 1, Soluble interleukin
receptors (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15),
Soluble TNF receptor, Somatomedin, Somatostatin, Somatotropin,
Streptokinase, Superantigens, i.e., Staphylococcal enterotoxins
(SEA, SEB, SEC1, SEC2, SEC3, SED, SEE), Superoxide dismutase (SOD),
Toxic shock syndrome toxin (TSST-1), Thymosin alpha 1, Tissue
plasminogen activator, Tumor necrosis factor beta (TNF beta), Tumor
necrosis factor receptor (TNFR), Tumor necrosis factor-alpha (TNF
alpha), Vascular Endothelial Growth Factor (VEGEF), Urokinase and
many others.
[0242] One class of proteins that can be made using the
compositions and methods for in vivo incorporation and modification
of unnatural amino acids into phage-displayed proteins described
herein includes transcriptional modulators or a portion thereof.
Example transcriptional modulators include genes and
transcriptional modulator proteins that modulate cell growth,
differentiation, regulation, or the like. Transcriptional
modulators are found in prokaryotes, viruses, and eukaryotes,
including fungi, plants, yeasts, insects, and animals, including
mammals, providing a wide range of therapeutic targets. It will be
appreciated that expression and transcriptional activators regulate
transcription by many mechanisms, e.g., by binding to receptors,
stimulating a signal transduction cascade, regulating expression of
transcription factors, binding to promoters and enhancers, binding
to proteins that bind to promoters and enhancers, unwinding DNA,
splicing pre-mRNA, polyadenylating RNA, and degrading RNA.
[0243] One class of proteins of the invention (e.g., proteins with
one or more unnatural amino acids) include biologically active
proteins such as cytokines, inflammatory molecules, growth factors,
their receptors, and oncogene products, e.g., interleukins (e.g.,
IL-1, IL-2, IL-8, etc.), interferons, FGF, IGF-I, IGF-II, FGF,
PDGF, TNF, TGF-.alpha., TGF-.beta., EGF, KGF, SCF/c-Kit, CD40/CD40,
VLA-4VCAM-1, ICAM-1/LFA-1, and hyalurin/CD44; signal transduction
molecules and corresponding oncogene products, e.g., Mos, Ras, Raf,
and Met; and transcriptional activators and suppressors, e.g., p53,
Tat, Fos, Myc, Jun, Myb, Rel, and steroid hormone receptors such as
those for estrogen, progesterone, testosterone, aldosterone, the
LDL receptor ligand and corticosterone.
[0244] Enzymes (e.g., industrial enzymes) or portions thereof with
at least one unnatural amino acid are also provided by the
invention. Examples of enzymes include, but are not limited to,
e.g., amidases, amino acid racemases, acylases, dehalogenases,
dioxygenases, diarylpropane peroxidases, epimerases, epoxide
hydrolases, esterases, isomerases, kinases, glucose isomerases,
glycosidases, glycosyl transferases, haloperoxidases,
monooxygenases (e.g., p450s), lipases, lignin peroxidases, nitrile
hydratases, nitrilases, proteases, phosphatases, subtilisins,
transaminase, and nucleases.
[0245] Many of these proteins are commercially available (See,
e.g., the Sigma BioSciences catalogue), and the corresponding
protein sequences and genes and, typically, many variants thereof,
are well-known (see, e.g., Genbank). Any of them can be modified by
the insertion of one or more unnatural amino acid according to the
invention, e.g., to alter the protein with respect to one or more
therapeutic, diagnostic or enzymatic properties of interest.
Examples of therapeutically relevant properties include serum
half-life, shelf half-life, stability, immunogenicity, therapeutic
activity, detectability (e.g., by the inclusion of reporter groups
(e.g., labels or label binding sites) in the unnatural amino
acids), reduction of LD.sub.50 or other side effects, ability to
enter the body through the gastric tract (e.g., oral availability),
or the like. Examples of diagnostic properties include shelf
half-life, stability, diagnostic activity, detectability, or the
like. Examples of relevant enzymatic properties include shelf
half-life, stability, enzymatic activity, production capability, or
the like.
[0246] A variety of other proteins can also be modified to include
one or more unnatural amino acid using compositions and methods of
the invention. For example, the invention can include substituting
one or more natural amino acids in one or more vaccine proteins
with an unnatural amino acid, e.g., in proteins from infectious
fungi, e.g., Aspergillus, Candida species; bacteria, particularly
E. coli, which serves a model for pathogenic bacteria, as well as
medically important bacteria such as Staphylococci (e.g., aureus),
or Streptococci (e.g., pneumoniae); protozoa such as sporozoa
(e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates
(Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such
as (+) RNA viruses (examples include Poxviruses e.g., vaccinia;
Picornaviruses, e.g. polio; Togaviruses, e.g., rubella;
Flaviviruses, e.g., HCV; and Coronaviruses), (-) RNA viruses (e.g.,
Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV;
Orthomyxovimses, e.g., influenza; Bunyaviruses; and Arenaviruses),
dsDNA viruses (Reoviruses, for example), RNA to DNA viruses, i.e.,
Retroviruses, e.g., HIV and HTLV, and certain DNA to RNA viruses
such as Hepatitis B.
[0247] Agriculturally related proteins such as insect resistance
proteins (e.g., the Cry proteins), starch and lipid production
enzymes, plant and insect toxins, toxin-resistance proteins,
Mycotoxin detoxification proteins, plant growth enzymes (e.g.,
Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase, "RUBISCO"),
lipoxygenase (LOX), and Phosphoenolpyruvate (PEP) carboxylase are
also suitable targets for unnatural amino acid modification.
[0248] In certain embodiments, the modified phage-displayed protein
of interest (or portion thereof) is encoded by a nucleic acid.
Typically, the nucleic acid comprises at least one selector codon,
at least two selector codons, at least three selector codons, at
least four selector codons, at least five selector codons, at least
six selector codons, at least seven selector codons, at least eight
selector codons, at least nine selector codons, ten or more
selector codons.
[0249] Genes coding for proteins or polypeptides of interest can be
mutagenized using methods well-known to one of skill in the art and
described herein under "Mutagenesis and Other Molecular Biology
Techniques" to include, e.g., one or more selector codon for the
incorporation of an unnatural amino acid. For example, a nucleic
acid for a protein of interest is mutagenized to include one or
more selector codon, providing for the insertion of the one or more
unnatural amino acids. The invention includes any such variant,
e.g., mutant, versions of any protein, e.g., including at least one
unnatural amino acid. Similarly, the invention also includes
corresponding nucleic acids, i.e., any nucleic acid with one or
more selector codon that encodes one or more unnatural amino
acid.
[0250] To make a phage-displayed protein that includes a
post-translationally modified unnatural amino acid, one can use
host cells and organisms that are adapted for the in vivo
incorporation of the unnatural amino acid via orthogonal tRNA/RS
pairs. Host cells are genetically engineered (e.g., transformed,
transduced or transfected) with one or more vectors that express
the orthogonal tRNA, the orthogonal tRNA synthetase, and a vector
that encodes the protein to be derivatized. Each of these
components can be on the same vector, or each can be on a separate
vector, or two components can be on one vector and the third
component on a second vector. The vector can be, for example, in
the form of a plasmid, a bacterium, a virus, a naked
polynucleotide, or a conjugated polynucleotide.
Defining Polypeptides by Immunoreactivity
[0251] Because the polypeptides of the invention provide a variety
of new polypeptide sequences (e.g., polypeptides comprising
unnatural amino acids in the case of proteins synthesized in the
translation systems herein, or, e.g., in the case of the novel
synthetases, novel sequences of standard amino acids), the
polypeptides also provide new structural features which can be
recognized, e.g., in immunological assays. The generation of
antisera, which specifically bind the polypeptides of the
invention, as well as the polypeptides which are bound by such
antisera, are a feature of the invention. The term "antibody," as
used herein, includes, but is not limited to a polypeptide
substantially encoded by an immunoglobulin gene or immunoglobulin
genes, or fragments thereof which specifically bind and recognize
an analyte (antigen). Examples include polyclonal, monoclonal,
chimeric, and single chain antibodies, and the like. Fragments of
immunoglobulins, including Fab fragments and fragments produced by
an expression library, including phage display, are also included
in the term "antibody" as used herein. See, e.g., Paul, Fundamental
Immunology, 4th Ed., 1999, Raven Press, New York, for antibody
structure and terminology.
[0252] In order to produce antisera for use in an immunoassay, one
or more of the immunogenic polypeptides is produced and purified as
described herein. For example, recombinant protein can be produced
in a recombinant cell. An inbred strain of mice (used in this assay
because results are more reproducible due to the virtual genetic
identity of the mice) is immunized with the immunogenic protein(s)
in combination with a standard adjuvant, such as Freund's adjuvant,
and a standard mouse immunization protocol (see, e.g., Harlow and
Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor
Publications, New York, for a standard description of antibody
generation, immunoassay formats and conditions that can be used to
determine specific immunoreactivity. Additional details on
proteins, antibodies, antisera, etc. can be found in International
Publication Numbers WO 2004/094593, entitled "EXPANDING THE
EUKARYOTIC GENETIC CODE;" WO 2002/085923, entitled "IN VIVO
INCORPORATION OF UNNATURAL AMINO ACIDS;" WO 2004/035605, entitled
"GLYCOPROTEIN SYNTHESIS;" and WO 2004/058946, entitled "PROTEIN
ARRAYS."
Photoregulation and Photocaging
[0253] The invention provides phage having displayed polypeptides
comprising at least one unnatural amino acid that is
post-translationally modified. The-posttranslational modification
can result in the attachment of any desired moiety onto the capsid
fusion polypeptide (and consequently onto the phage). In some
embodiments, the conjugated moiety that is coupled to the unnatural
amino acid is photoregulated, thereby producing a photoregulated
modified unnatural amino acid.
[0254] Photoregulated amino acids (e.g., photochromic,
photocleavable, photoisomerizable, etc.) can be used to spatially
and temporally control a variety of biological process, e.g., by
directly regulating the activity of enzymes, receptors, ion
channels or the like, or by modulating the intracellular
concentrations of various signaling molecules. See, e.g., Shigeri
et al., Pharmacol. Therapeut., 2001, 91:85; Curley, et al.,
Pharmacol. Therapeut., 1999, 82:347; Curley, et al., Curr. Op.
Chem. Bio., 1999, 3:84; "Caged Compounds" Methods in Enzymology,
Marriott, G., Ed, Academic Press, NY, 1998, V. 291; Adams, et al.,
Annu. Rev. Physiol., 1993, 55:755+; and Bochet, et al., J. Chem.
Soc., Perkin 1, 2002, 125. In various embodiments herein, the
compositions and methods comprise photoregulated amino acids.
[0255] "Photoregulated amino acids" are typically, e.g.,
photosensitive amino acids. Photoregulated amino acids in general
are those that are controlled in some fashion by light (e.g., UV,
IR, etc.). Thus, for example, if a photoregulated amino acid is
incorporated into a polypeptide having biological activity,
illumination can alter the amino acid, thereby changing the
biological activity of the peptide. Some photoregulated amino acids
can comprise "photocaged amino acids," "photosensitive amino
acids," "photolabile amino acids," "photoisomerizable," etc. "Caged
species," such as caged amino acids, or caged peptides, are those
trapped inside a larger entity (e.g., molecule) and that are
released upon specific illumination. See, e.g., Adams, et al.,
Annu. Rev. Physiol., 1993, 55:755-784. "Caging" groups of amino
acids can inhibit or conceal (e.g., by disrupting bonds which would
usually stabilize interactions with target molecules, by changing
the hydrophobicity or ionic character of a particular side chain,
or by steric hindrance, etc.) biological activity in a molecule,
e.g., a peptide comprising such amino acid. "Photoisomerizable"
amino acids can switch isomer forms due to light exposure. The
different isomers of such amino acids can end up having different
interactions with other side chains in a protein upon
incorporation. Photoregulated amino acids can thus control the
biological activity (either through activation, partial activation,
inactivation, partial inactivation, modified activation, etc.) of
the peptides in which they are present. See Adams above and other
references in this section for further definitions and examples of
photoregulated amino acids and molecules.
[0256] A number of photoregulated amino acids are known to those in
the art and many are available commercially. Methods of attaching
and/or associating photoregulating moieties to amino acids are also
known. Such photoregulated amino acids in general are amenable to
various embodiments herein. It will be appreciated that while a
number of possible photoregulating moieties, e.g., photocaging
groups and the like, as well as a number of photoregulated amino
acids are listed herein, such recitation should not be taken as
limiting. Thus, the current invention is also amenable to
photoregulating moieties and photoregulated amino acids that are
not specifically recited herein.
[0257] As stated, a number of methods are optionally applicable to
create a photoregulated amino acid. Thus, for example, a
photoregulated amino acid, e.g., a photocaged amino acid can be
created by protecting its a-amino group with compounds such as BOC
(butyloxycarbonyl), and protecting the a-carboxyl group with
compounds such as a t-butyl ester. Such protection can be followed
by reaction of the amino acid side chain with a photolabile caging
group such as 2-nitrobenzyl, in a reactive form such as
2-nitrobenzylchloroformate, .alpha.-carboxyl 2-nitrobenzyl bromide
methyl ester, or 2-nitrobenzyl diazoethane. After the photolabile
cage group is added, the protecting groups can be removed via
standard procedures. See, e.g., U.S. Pat. No. 5,998,580.
[0258] As another example, lysine residues can be caged using
2-nitrobenzylchloroformate to derivatize the .epsilon.-lysine amino
group, thus eliminating the positive charge. Alternatively, lysine
can be caged by introducing a negative charge into a peptide (which
has such lysine) by use of an .alpha.-carboxy
2-nitrobenzyloxycarbonyl caging group. Additionally, phosphoserine
and phosphothreonine can be caged by treatment of the phosphoamino
acid or the phosphopeptide with 1(2-nitrophenyl)diazoethane. See,
e.g., Walker et al., Meth Enzymol. 172:288-301, 1989. A number of
other amino acids are also easily amenable to standard caging
chemistry, for example serine, threonine, histidine, glutamine,
asparagine, aspartic acid and glutamic acid. See, e.g., Wilcox et
al., J. Org. Chem. 55:1585-1589, 1990). Again, it will be
appreciated that recitation of particular photoregulated (amino
acids and/or those capable of being converted to photoregulated
forms) should not necessarily be taken as limiting.
[0259] Amino acid residues can also be made photoregulated (e.g.,
photosensitive or photolabile) in other fashions. For example,
certain amino acid residues can be created wherein irradiation
causes cleavage of a peptide backbone that has the particular amino
acid residue. For example a photolabile glycine, 2-nitrophenyl
glycine, can function in such a manner. See, e.g., Davis, et al.,
1973, J. Med. Chem., 16:1043-1045. Irradiation of peptides
containing 2-nitrophenylglycine will cleave the peptide backbone
between the alpha carbon and the alpha amino group of
2-nitrophenylglycine. Such cleavage strategy is generally
applicable to amino acids other than glycine, if the 2-nitrobenzyl
group is inserted between the alpha carbon and the alpha amino
group.
[0260] A large number of photoregulating groups, e.g., caging
groups, and a number of reactive compounds used to covalently
attach such groups to other molecules such as amino acids, are well
known in the art. Examples of photoregulating (e.g., photolabile,
caging) groups include, but are not limited to:
o-nitrobenzyl-serine, O-(2-nitrobenzyl)-L-tyrosine, nitroindolines;
N-acyl-7-nitroindolines; phenacyls; hydroxyphenacyl; brominated
7-hydroxycoumarin-4-ylmethyls (e.g., Bhc); benzoin esters;
dimethoxybenzoin; meta-phenols; 2-nitrobenzyl;
1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE);
4,5-dimethoxy-2-nitrobenzyl (DMNB); alpha-carboxy-2-nitrobenzyl
(CNB); 1-(2-nitrophenyl)ethyl (NPE); 5-carboxymethoxy-2-nitrobenzyl
(CMNB); (5-carboxymethoxy-2-nitrobenzyl)oxy) carbonyl;
(4,5-dimethoxy-2-nitrobenzyl)oxy) carbonyl; desoxybenzoinyl; and
the like. See, e.g., U.S. Pat. No. 5,635,608 to Haugland and Gee
(Jun. 3, 1997) entitled ".alpha.-carboxy caged compounds" Neuro 19,
465 (1997); J Physiol 508.3, 801 (1998); Proc Natl Acad Sci USA
1988 September, 85(17):6571-5; J Biol Chem 1997 Feb. 14,
272(7):4172-8; Neuron 20, 619-624, 1998; Nature Genetics, vol.
28:2001:317-325; Nature, vol. 392,1998:936-941; Pan, P., and
Bayley, H. "Caged cysteine and thiophosphoryl peptides" FEBS
Letters 405:81-85 (1997); Pettit et al. (1997) "Chemical two-photon
uncaging: a novel approach to mapping glutamate receptors" Neuron
19:465-471; Furuta et al. (1999) "Brominated
7-hydroxycoumarin-4-ylmethyls: novel photolabile protecting groups
with biologically useful cross-sections for two photon photolysis"
Proc. Natl. Acad. Sci. 96(4): 1193-1200; Zou et al. "Catalytic
subunit of protein kinase A caged at the activating
phosphothreonine" J. Amer. Chem. Soc. (2002) 124:8220-8229; Zou et
al. "Caged Thiophosphotyrosine Peptides" Angew. Chem. Int. Ed.
(2001) 40:3049-3051; Conrad II et al. "p-Hydroxyphenacyl
Phototriggers: The reactive Excited State of Phosphate
Photorelease" J. Am. Chem. Soc. (2000) 122:9346-9347; Conrad II et
al. "New Phototriggers 10: Extending the .pi.,.pi.* Absorption to
Release Peptides in Biological Media" Org. Lett. (2000)
2:1545-1547; Givens et al. "A New Phototriggers 9:
p-Hydroxyphenacyl as a C-Terminus Photoremovable Protecting Group
for Oligopeptides" J. Am. Chem. Soc. (2000) 122:2687-2697; Bishop
et al. "40-Aminomethyl-2,20-bipyridyl-4-carboxylic Acid (Abc) and
Related Derivatives: Novel Bipyridine Amino Acids for the
Solid-Phase Incorporation of a Metal Coordination Site Within a
Peptide Backbone" Tetrahedron (2000) 56:4629-4638; Ching et al.
"Polymers As Surface-Based Tethers with Photolytic triggers
Enabling Laser-Induced Release/Desorption of Covalently Bound
Molecules" Bioconjugate Chemistry (1996) 7:525-8; BioProbes
Handbook, 2002 from Molecular Probes, Inc.; and Handbook of
Fluorescent Probes and Research Products, Ninth Edition or Web
Edition, from Molecular Probes, Inc, as well as the references
herein. Many compounds, kits, etc. for use in caging various
molecules are commercially available, e.g., from Molecular Probes,
Inc. Additional references are found in, e.g., Merrifield, Science
232:341 (1986) and Corrie, J. E. T. and Trentham, D. R. (1993) In:
Biological Applications of Photochemical Switches, ed., Morrison,
H., John Wiley and Sons, Inc. New York, pp. 243-305. Examples of
suitable photosensitive caging groups include, but are not limited
to, 2-nitrobenzyl, benzoin esters, N-acyl-7-nitindolines,
meta-phenols, and phenacyls.
[0261] In some embodiments, a photoregulating (e.g., caging) group
can optionally comprise a first binding moiety, which can bind to a
second binding moiety. For example, a commercially available caged
phosphorarnidite
[1-N-(4,4'-Dimethoxytrityl)-5-(6-biotinamidocaproamidomethyl)-
1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite
(PC Biotin Phosphoramadite, from Glen Research Corp.) comprises a
photolabile group and a biotin (the first binding moiety). A second
binding moiety, e.g., streptavidin or avidin, can thus be bound to
the caging group, increasing its bulkiness and its effectiveness at
caging. In certain embodiments, a caged component comprises two or
more caging groups each comprising a first binding moiety, and the
second binding moiety can bind two or more first binding moieties
simultaneously. For example, the caged component can comprise at
least two biotinylated caging groups; binding of streptavidin to
multiple biotin moieties on multiple caged component molecules
links the caged components into a large network. Cleavage of the
photolabile group attaching the biotin to the component results in
dissociation of the network.
[0262] Traditional methods of creating caged polypeptides
(including e.g. peptide substrates and proteins such as antibodies
or transcription factors) include, e.g., by reacting a polypeptide
with a caging compound or by incorporating a caged amino acid
during synthesis of a polypeptide. See, e.g., U.S. Pat. No.
5,998,580 to Fay et al. (Dec. 7, 1999) entitled "Photosensitive
caged macromolecules"; Kossel et al. (2001) PNAS 98:14702-14707;
Trends Plant Sci (1999) 4:330-334; PNAS (1998) 95:1568-1573; J. Am.
Chem. Soc. (2002) 124:8220-8229; Pharmacology & Therapeutics
(2001) 91:85-92; and Angew. Chem. Int. Ed. Engl. (2001)
40:3049-3051. A photolabile polypeptide linker (e.g., for
connecting a protein transduction domain and a sensor, or the like)
can, for example, comprise a photolabile amino acid such as that
described in U.S. Pat. No. 5,998,580.
[0263] Irradiation with light can, e.g., release a side chain
residue of an amino acid that is important for activity of the
peptide comprising such amino acid. Additionally, in some
embodiments, uncaged amino acids can cleave the peptide backbone of
the peptide comprising the amino acid and can thus, e.g., open a
cyclic peptide to a linear peptide with different biological
properties, etc.
[0264] Activation of a caged peptide can be done through
destruction of a photosensitive caging group on a photoregulated
amino acid by any standard method known to those skilled in the
art. For example, a photosensitive amino acid can be uncaged or
activated by exposure to a suitable conventional light source, such
as lasers (e.g., emitting in the UV range or infrared range). Those
of skill in the art will be aware of and familiar with a number of
additional lasers of appropriate wavelengths and energies as well
as appropriate application protocols (e.g., exposure duration,
etc.) that are applicable to use with photoregulated amino acids
such as those utilized herein. Release of photoregulated caged
amino acids allows control of the peptides that comprise such amino
acids. Such control can be both in terms of location and in terms
of time. For example, focused laser exposure can uncage amino acids
in one location, while not uncaging amino acids in other
locations.
[0265] Those skilled in the art will appreciate a variety of assays
can be used for evaluating the activity of a photoregulated amino
acid, e.g., the assays described in the examples herein. A wide
range of, e.g., cellular function, tissue function, etc. can be
assayed before and after the introduction of a peptide comprising a
photoregulated amino acid into the cell or tissue as well as after
the release of the photoregulated molecule.
[0266] The compositions and methods herein can be utilized in a
number of aspects. For example, photoregulated amino acids (e.g.,
in peptides) can deliver therapeutic compositions to discrete
locations of a body since the release or
activation/deactivation/etc. of the photoregulated amino acid can
be localized through targeted light exposure, etc. It will also be
appreciated that the methods, structures, and compositions of the
invention are applicable to incorporation/use of photoregulated
natural amino acids (e.g., ones with photoregulating moieties
attached/associated with them).
[0267] Photochromic and photocleavable groups can be used to
spatially and temporally control a variety of biological processes,
either by directly regulating the activity of enzymes (see, e.g.,
Westmark, et al., J. Am. Chem. Soc. 1993, 115:3416-19 and Hohsaka,
et al., J. Am. Chem. Soc. 1994, 116:413-4), receptors (see, e.g.,
Bartels, et al., Proc. Natl. Acad. Sci. USA, 1971, 68:1820-3;
Lester, et al., Nature 1977, 266:373-4: Cruz, et al., J. Am. Chem.
Soc., 2000, 122:8777-8; and, Pollitt, et al., Angew. Chem. Int. Ed.
Engl., 1998, 37:2104-7), or ion channels (see, e.g., Lien, et al.,
J. Am. Chem. Soc. 1996, 118:12222-3; Borisenko, et al., J. Am.
Chem. Soc. 2000, 122:6364-70; and, Banghart, et al., Nat. Neurosci.
2004, 7:1381-6.), or by modulating the intracellular concentrations
of various signaling molecules (see, e.g., Adams, et al., Annu.
Rev. Physiol. 1993, 55:755-84). In general, this requires the
chemical modification of either a protein or small molecule with a
photoreactive ligand such as azobenzene or a nitrobenzyl group. The
ability to genetically incorporate photoresponsive amino acids into
proteins at defined sites directly in living organisms would
significantly extend the scope of this technique. See, e.g., Wu, et
al., J. Am. Chem. Soc. 2004, 126:14306-7.
Kits
[0268] Kits are also a feature of the invention. For example, a kit
for producing a phage having a displayed polypeptide comprising at
least one unnatural amino acid that is post-translationally
modified is a feature of the invention. For example, such kits can
comprise various components selected from: a container to hold the
kit components, instructional materials for producing the modified
phage, a nucleic acid comprising the phage genomic material,
nucleic acid comprising a polynucleotide sequence encoding an
O-tRNA, nucleic acid comprising a polynucleotide encoding an O-RS,
an unnatural amino acid, for example an aryl-azide amino acid
(e.g., para-azido-L-phenylalanine) or an alkynyl-amino acid (e.g.,
para-propargyloxyphenylalanine), reagents for the
post-translational modification of the unnatural amino acid (e.g.,
reagents for the Staudinger ligation or the [3+2] cycloaddition
reaction), and a suitable strain of E. coli host cells for
expression of the O-tRNA/O-RS and production of the phage.
EXAMPLES
[0269] The following examples are offered to illustrate, but not to
limit the claimed invention. One of skill will recognize a variety
of non-critical parameters that may be altered without departing
from the scope of the claimed invention.
Example 1
The Generation of Phage Displayed Polypeptides Comprising Unnatural
Amino Acids
[0270] The present Example describes compositions and methods for
the generation of phage displayed polypeptides comprising unnatural
amino acids. As described previously, orthogonal translation
components can be used in suitable host cells to selectively
introduce any of a large number of unnatural amino acids into
proteins in vivo with good efficiency and high fidelity. As
described herein, these orthogonal translation components and
methodologies can be adapted for use in phage display systems as a
general approach to the generation of phage-displayed polypeptide
libraries containing unnatural amino acid building blocks.
[0271] Two plasmids, pDULE/CM and M13KE, were used to generate
phage that display polypeptides containing unnatural amino acids as
fusions to the pIII protein of the M13 filamentous phage. Plasmid
pDULE/CM, which has a p15A origin, constitutively expresses a
Methanococcus jannaschii amber suppressor tRNATyr (MjtRNA) and a
mutant M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS; synthetase
variant clone number 7 as described in Chin et al., J. Am. Chem.
Soc., (2002) 124:9026-9027; see also FIG. 2 and SEQ ID NO: 10) in
Escherichia coli. This mutant MjTyrRS aminoacylates the amber
suppressor tRNA (e.g., the orthogonal tRNA shown in FIG. 1; SEQ ID
NO: 1) with the desired unnatural amino acid (e.g., the amino acids
shown in FIG. 3). Growth of E. coli Top 10 F' harboring pDULE/CM
(designated strain TTS) in the presence of the corresponding
unnatural amino acid results in the incorporation of the unnatural
amino acid at the site specified by the amber codon TAG. The second
plasmid, M13KE, was a phage vector used for pentavalent N-terminal
pIII display; a derivative, pM13KE-SBP, displaying a pIII fusion
streptavidin binding peptide (SBP), AGXTLLAHPQ (SEQ ID NO: 11), was
used in this study. The N-terminal AG sequence facilitates cleavage
of the signal peptide. The third residue, X, encoded by amber
nonsense codon TAG, designates the unnatural amino acid to be
incorporated. Expression of the pIII fusion protein in E. coli
strain TTS in the presence of the unnatural amino acid should
afford viable phage that display the peptide containing the
unnatural amino acid as a pIII fusion. To prepare the initial phage
stocks, plasmid pM13KE-SBP was transformed into the E. coli strain
XL1-Blue, a natural amber suppression strain that incorporates
glutamine at residue X.
[0272] To examine the dependence of phage plaque formation on the
presence of the unnatural amino acid, M13KE-SBP phage and M13KE
wild-type phages were plated on E. coli strain TTS/RS 3 cell lawns
(where RS 3 designates an aminoacyl tRNA synthetase specific for
p-acetylphenylalanine, structure 3 in FIG. 3) in the presence and
absence of 2 mM p-acetylphenylalanine 3. In the presence of the
unnatural amino acid, both M13KE-SBP phage and M13KE wild-type
phage formed normal-sized plaques after overnight incubation at
37.degree. C. However, in the absence of the unnatural amino acid,
only M13KE wild-type phage formed plaques. No plaque formation was
observable for M13KE-SBP phage in the absence of
p-acetyl-phenylalanine. The M13KE-SBP phage yield in the natural
glutaminyl amber suppressor strain XL1-Blue was 2.times.10.sup.11
plaque-forming units per milliliter of culture (PFU/mL). The yield
of M13KE-SBP phage in E. coli TTS/RS 3 in the presence of 2 mM
p-acetylphenylalanine 3 is comparable to that produced in XL1-Blue
and is dependent on the presence of p-acetylphenylalanine 3. In the
presence of this unnatural amino acid, the phage yield is
1.8.times.10.sup.11 PFU/mL; in the absence of the unnatural amino
acid the phage yield is reduced by 81-fold (see FIG. 4). In a
large-scale phage preparation, a difference in yield of over
1000-fold was obtained. The phage yield experiments were carried
out with five E. coli TTS/RS cell lines that incorporate five
distinct unnatural amino acids. These were: O-methyltyrosine 1,
p-azidophenylalanine 2, p-acetylphenylalanine 3,
p-benzoylphenylalanine 4, and 3-(2-naphthyl)alanine 5 (see FIG. 3).
A similar dependence on the presence of the unnatural amino acid
for phage yield was observed in each system (see FIG. 4),
indicating that this phage-display scheme is likely to be general
for a large number of unnatural amino acids.
Example 2
Plasmid Constructions
[0273] The present Example describes the construction of the
plasmids used to expresses the orthogonal tRNA and orthogonal
aminoacyl tRNA synthetase in E. coli host cells.
Construction of Plasmid pDULE/CM
[0274] Plasmid pDULE with an ampicillin resistant marker was
digested with Bsm I and treated with Mung Bean nuclease to create a
blunt end. The resulting DNA was digested with Cla I and purified
by agarose gel electrophoresis. The chloramphenicol
acetyltransferase gene was amplified from plasmid pACYC184 (NEB) by
PCR using the primers: TABLE-US-00002 SEQ ID Primer Sequence NO:
FT18 5'-GACAGCTTATCATCGATGAGACGTTGATCGGCACGTA 12 AG FT19
5'-GGTTGGTTTGCGCATTCAGCGCTAACCGTTTTTATCA 13 GGC
[0275] The PCR product was digested with Cla I and ligated with the
pDULE to give pDULE/CM. The TTS cell line was generated by
transformation of plasmid pDULE/CM into Top 10 F' (Invitrogen).
Construction OF pM13KE-SBP PLASMID:
[0276] The streptavidin binding peptide pIII fusion was prepared
using extension TABLE-US-00003 primer FT121: (SEQ ID NO:14)
5'-CATGCCCGGGTACCTTTCTATTCTC and template FT126: (SEQ ID NO:15)
5'-CATGTTTCGGCCGAGCCCCCACCCTGCGGATGAGCCAGCAAAGTCTA
GCCGGCAGAGTGAGAATAGAAAGGTACCCGGG;
digested with Eag I and Acc65 I. Insertion of this fragment into
plasmid pM13KE (New England BioLabs) between the Eag I and Acc65 I
sites with T4 ligase yielded pM13KE-SBP. The ligation product was
transformed into XL1-Blue and plated on LB plates with an XL1-Blue
cell lawn in the presence of 20 .mu.g/mL IPTG and 20 .mu.g/mL XGal
to generate blue phage plaques.
Example 3
Phage Culture and Phage Titering Protocols
[0277] The present Example describes the general methodologies of
phage culture and titering as used herein.
General Phage Production Protocol in E. coli XL1-BLUE
[0278] A single phage plaque was added to 10 mL of 2.times.YT
containing mid-log E. coli strain XL1-Blue and 12 .mu.g/mL of
tetracycline. After incubation at 37.degree. C. for 5 hrs, the
culture was centrifuged at 6000.times. g, at 4.degree. C. for 5
min. Phage was precipitated from the supernatant with 20% volume
PEG buffer (20% PEG 8000, 2.5 M NaCl). The mixture was kept at
4.degree. C. overnight and centrifuged at 10,000.times. g for 10
min at 4.degree. C. The phage pellet was dissolved in 500 .mu.L of
1.times. PBS, pH 7.4, centrifuged at 20,000.times. g for 10 min at
4.degree. C. to remove the remaining cell debris, and stored at
4.degree. C.
General Phage Production Protocol in E. coli TTS
[0279] A single phage plaque was added to 10 ml of 2.times.YT
containing mid-log TTS and 12 .mu.g/mL of tetracycline, 34 .mu.g/mL
chloramphenicol and 2 mM of appropriate unnatural amino acid. The
rest of the protocol is the same as the general phage production
protocol in E. coli XL1-Blue.
General Plaque Formation and Phage Titer Experiment
[0280] After a series of 10 fold dilutions in microtiter plates, 5
.mu.l of both M13KE wild type and M13KE-SBP phages were plated on
LB Agar plates with a TTS/RS 3 cell lawn supplemented with 20
.mu.g/mL IPTG, 20 .mu.g/mL XGal, 12 .mu.g/mL tetracycline, 34
.mu.g/mL chloramphenicol and 2 mM of the corresponding unnatural
amino acid. The plates were incubated at 37.degree. C.
overnight.
Phage Titer
[0281] After a series of 10 fold dilutions in microtiter plates, 5
.mu.L of phage were plated on LB agar plates with an XL1-Blue cell
lawn supplemented with 20 .mu.g/mL IPTG, 20 .mu.g/mL XGal, and 12
.mu.g/mL tetracycline. The plates were incubated at 37.degree. C.
overnight.
Example 4
Covalent Conjugation of Phage Displayed Polypeptides Comprising
p-azido-L-phenylalanine using a [3+2] Cycloaddition Reaction
[0282] The present Example describes the covalent modification of a
phage-displayed polypeptide comprising p-azido-L-phenylalanine.
This covalent modification uses a [3+2] cycloaddition reaction to
conjugate an alkyne-containing moiety to the
p-azido-L-phenylalanine, resulting in a triazole linkage between
the polypeptide and the alkyne-containing moiety. FIG. 15 provides
the general reaction chemistry of the [3+2] cycloaddition
reaction.
[0283] M13KE-SBP phage were produced in E. coli TTS/RS 2 in the
presence of 2 mM p-azidophenylalanine 2. The resulting phage were
then conjugated with the alkyne-derivatized fluorescein dye shown
in FIG. 5, structure 6 (Deiters et al., J. Am. Chem. Soc. (2003)
125:11782-11783) by a highly specific azide-alkyne [3+2]
cycloaddition (Rostovtsev et al., Angew. Chem. Int. Ed. (2002)
41:2596-2599). Phage prepared in XL1-Blue were used as a negative
control.
[0284] Specifically, the cycloaddition conjugation reactions
between phage and alkyne derivatized fluorescein dye 6 were
conducted as follows. Phage from a stock solution (50 .mu.L, about
10.sup.11 PFU) was precipitated by PEG and dissolved in 90 .mu.L of
100 .mu.M potassium phosphate buffer (PB), pH 8.0. The phage
solution was supplemented with 5 .mu.L of tert-butanol, 2 mM
tris(carboxyethyl)phosphine (TCEP), 2 mM tris(triazolyl) amine
ligand, 2 mM fluorescein dye 6 and 1 mM CuSO.sub.4. The final
reaction volume was 100 .mu.L. Upon the addition of tris(triazolyl)
amine ligand, phage precipitated. The reaction mixture was
incubated at 4.degree. C. for 16 hours and was centrifuged at
20,000.times. g for 10 min. Phage precipitated completely under the
reported conditions [2 mM tris(carboxyethyl)phosphine (TCEP), 2 mM
tris-(triazolyl) amine ligand, 2 mM fluorescein dye 6 and 1 mM
CuSO4 in potassium phosphate buffer (PB) at pH 8.0 with 5%
tert-butyl alcohol as cosolvent] (see, Wang et al., J. Am. Chem.
Soc. (2003) 125:3192-3193). Replacement of TCEP with Cu wire
provided no improvement. Precipitation was minimized when diluted
reagents were used (0.1 mM TCEP, 0.2 mM ligand, 0.2 mM fluorescein
dye 6 and 0.1 mM CUSO.sub.4 in PB buffer at pH 8.0). See, Link and
Tirrell, J. Am. Chem. Soc. (2003) 125:11164-11165.
[0285] After conjugation, the reaction mixture was dialyzed and
subjected to SDS-PAGE and Western blot analysis (see FIG. 6). The
Western analysis used both anti-fluorescein (part I) and anti-pIII
(part II) primary antibodies to verify the identity of the
separated protein species. Specifically, both precipitant and
concentrated supernatant from the previous cycloaddition reaction
were electrophoresed by 4-20% SDS-PAGE gel (Invitrogen) and
transferred to a nitrocellulose membrane (semidry 20 V, 20 min).
The membrane was blocked with 5% skim milk in 1.times.PBS at
4.degree. C. overnight and incubated with a 1/1000 dilution of
anti-fluorescein rabbit IgG (Molecular Probes) at room temperature
for 2 hours. The membrane was washed and then probed with 1/10,000
dilution of anti-rabbit mouse IgG-alkaline phosphatase conjugate
(Sigma). The membrane was washed six times with PBST (PBS, 0.5%
Tween-20), developed with ECF (Amersham Biosciences) and scanned
using a phosphor imager. An anti-pIII western analysis was also
conducted, where anti-pIII mouse IgG (MoBiTec) was used as the
primary antibody. Anti-mouse AP conjugate was used as the secondary
antibody.
[0286] Development of these blots (see FIG. 6, part I) revealed
that the fluorescein conjugate was detected as a single band only
in the case of phage produced in TTS/RS 2 supplemented with 2 mM
p-azidophenylalanine 2 (lane a), in contrast to phage produced in
XL-1 Blue E. coli (lane b). The identity or this band was further
confirmed as the pIII minor coat protein by the anti-pE Western
blot analysis (part II). These results demonstrate that the
unnatural amino acid is incorporated specifically into the pm coat
protein of the unnatural phage.
Example 5
Preservation of Biological Activity of a Phage Displayed
Polypeptide Comprising the Unnatural Amino Acid
p-azido-L-phenylalanine
[0287] The present Example illustrates the preservation of
biological activity of the phage-displayed streptavidin binding
peptide (SBP) fusion comprising p-azido-L-phenylalanine. The
streptavidin binding activity was assayed in the phage
preparations.
[0288] To demonstrate that the mutant streptavidin binding peptide
presented on the N-terminus of the pIII protein is functional, a
phage-binding enzyme-linked immunosorbent assay (ELISA) was
utilized. This system used M13KE-SBP phage prepared in TTS/RS 2 and
TTS/RS 3 cells supplemented with 2 mM p-azidophenylalanine 2 or
p-acetylphenylalanine 3, respectively (see the data in FIG. 7 and
corresponding graph in FIG. 8). M13KE-SBP phage prepared in
XL1-Blue served as a positive control, while the wild-type M13KE
phage served as a negative control.
[0289] In this analysis, streptavidin coated microtiter plates
(Pierce) were blocked with 300 .mu.L of either 4% BSA in
1.times.PBS, or 4% BSA in 1.times.PBS with 10 .mu.M biotin at
4.degree. C. overnight. Following washing, 100 .mu.L of either
M13KE phage (10.sup.11 PFU), M13KE-SBP phage prepared in E. coli
XL1-Blue, M13KE-SBP prepared in TTS/RS 2 or TTS/RS 3 with 2 mM of
the corresponding unnatural amino acid were added to the wells with
four-fold serial dilution. After incubation at room temperature for
two hours, the wells were washed with 200 .mu.L PBST three times
and incubated with a 1/10,000 dilution of anti-M13 HRP conjugate
(Amersham Biosciences) for one hour. Following ten washes with 200
.mu.L PBST, the wells were developed with 100 .mu.L OPD substrate
in stable peroxidase buffer (Pierce). The reaction was terminated
by addition of 100 .mu.L 2.5 N H.sub.2SO.sub.4. The OD.sub.492 nm
of each well was recorded with a plate reader (see FIG. 7). The
value of each point in the table shown in FIG. 7 is the average of
three experiments. The error is less than 10%.
[0290] This data is shown graphically in FIG. 8. This figure shows
that M13KE-SBP phage prepared in TTS/p-azidophenylalanine 2 and
TTS/p-acetylphenylalanine 3 bind to streptavidin more strongly than
the positive control phage prepared in XL1-Blue. This increase in
observed affinity might result from increased binding affinity or
proteolytic stability of the displayed peptide containing the
unnatural amino acids.
[0291] In a model phage selection experiment, wild-type phage and
phage carrying the mutant SBP with p-azido-L-phenylalanine were
exposed to streptavidin coated wells, followed by recovery of the
bound phage. Titer of the recovered enriched phage following
elution was used as a measure of the binding specificity of the
page for the streptavidin.
[0292] Specifically, two wells from a streptavidin coated
microtiter plate (Pierce) were blocked with 300 .mu.L of 4% BSA in
1.times.PBS at 4.degree. C. overnight. After washing, separate
streptavidin coated wells were incubated with similar numbers of
phage (each in 100 .mu.L) of either M13KE-SBP prepared in TTS/RS 3
or M13KE wild type phage at room temperature for 2 hours and washed
10 times with 200 .mu.L PBST. The bound phage was eluted from the
plate solid support with 10 .mu.M biotin 1.times.PBS, pH 7.4. The
input and output phage were titered (see, FIG. 9).
[0293] The recovery rate of M13KE-SBP phage prepared in TTS/RS 3 is
9.times.10.sup.3 fold over that of M13KE wild-type phage (FIG. 9).
These experimental results show that the mutant SBP is displayed on
phage and is functional (i.e., retains the ability to specifically
bind strepavidin).
[0294] The generalization of phage display to include unnatural
amino acids should significantly increases the scope of phage
display technology. For example, the incorporation unnatural amino
acids into phage-displayed polypeptides can lead to increased
binding affinity and specificity, conformationally constrained
backbones and side chains, and enhanced proteolytic stability.
Unnatural amino acids can provide reactive sites for the
conjugation of nonpeptidic molecules as well as photoaffinity
labels for the identification of orphan ligands or receptors.
Finally, this methodology is also applicable to other display
formats such as ribosome and yeast display.
Example 6
Selective Covalent Modification of Phage Displayed Polypeptides
Comprising p-azido-L-phenylalanine Using the Staudinger Ligation
Reaction
[0295] The present Example illustrates the selective covalent
modification of phage-displayed polypeptides comprising
p-azido-L-phenylalanine using the Staudinger ligation reaction.
This Example used the phage-displayed streptavidin binding peptide
(SBP)/pIII fusion protein described in Example 1 as the substrates
for the Staudinger reaction, as illustrated in FIG. 10.
[0296] As described in Examples 1-3, a phage display system was
created in which the streptavidin binding peptide (SBP), AGXTLLAHPQ
(SEQ ID NO: 11), was displayed pentavalently as a fusion to the
pIII protein of M13 filamentous phage. The N-terminal AG sequence
facilitates cleavage of the signal peptide; the third residue, X,
encoded by the amber nonsense codon TAG, designates the unnatural
amino acid to be incorporated. The phage Ph-Az (encoding SBP with
p-azido-L-phenylalanine 2 at residue X) was prepared in E. coli
strain TTS/RS in the presence of 2 mM p-azido-L-phenylalanine 2
with good efficiency and high fidelity. E. coli TTS/RS contains a
plasmid that constitutively expresses a Methanococcus jannaschii
mutant amber suppressor tRNA.sub.CUA.sup.Tyr
(mutRNA.sub.CUA.sup.Tyr) and a mutant M. jannaschii tyrosyl-tRNA
synthetase (MjTyrRS) which specifically charges
mutRNA.sub.CUA.sup.Tyr with p-azido-L-phenylalanine 2. As a
negative control, another SBP displayed phage (Ph-Q) was prepared
in E. coli XL1-Blue, a natural amber suppression strain that
incorporates glutamine at residue X.
[0297] Using this phage system, the feasibility of using phage
displayed polypeptides comprising the unnatural amino acid
p-azido-L-phenylalanine 2 as a substrate for a selective Staudinger
modification was examined. The fluorescein-derived phosphines 7 and
8 (see the structures in FIG. 10) were used for the Staudinger
ligation reaction since they can be easily detected.
[0298] Compound 7 was synthesized following published procedures
(Saxon and Bertozzi, Science 2000, 287, 2007-2010; Wang et al.,
Bioconjugate Chem. 2003, 14, 697-701). Compound 8 was prepared by
the coupling reaction of 2-(diphenylphosphino)phenol (74 mg, 0.27
mmol), see Suarez et al., Organometallics 2002, 21, 4611-4621, and
5(6)-carboxyfluorescein (100 mg, 0.27 mmol) in the presence of
dicyclohexylcarbodiimide (62 mg, 0.3 mmol) in anhydrous DMF (1 mL)
at ambient temperature for 12 hrs, and purified using preparative
TLC as a red powder (3 mg, 2%); HRMS (ESI-TOF):
C.sub.39H.sub.24O.sub.7P.sub.1 [M-1].sup.-calcd: 635.1265; found
635.1248.
[0299] According to the scheme in FIG. 10, phosphine 7 can react
with phage Ph-Az to form an aza-ylide intermediate (Staudinger and
Meyer, Helv. Chim. Acta 1919, 2, 635-646), followed by
intramolecular cyclization (Saxon and Bertozzi, Science 2000, 287,
2007-2010) to ultimately yield a fluorescein labeled phage product.
The conjugation of 8 with Ph-Az should undergo a traceless
Staudinger ligation by a similar reaction mechanism to yield a
fluorescein-labeled phage without an intervening triphenylphosphine
oxide group.
[0300] Conjugation reactions between the phage molecules and the
phosphines 7 and 8 were carried out between phage Ph-Az and
triphenylphosphines 7 and 8 with approximately 10.sup.11 phage
particles and 0.01 mM phosphine in 10 mM phosphate buffered saline
solution (PBS, pH 7.4); similar reactions were carried out with
phage Ph-Q as a negative control. A stock (0.5 mM) of each
phosphine reactant was prepared in DMF, and was diluted with
reaction buffer to a final concentration of 0.01 mM and total
volume of 50 .mu.L. The ligation reactions were carried out at
ambient temperature with shaking for 16 hrs. The reaction mixture
was then dialyzed against PBS and subjected to subsequent
analysis.
[0301] The ligation reaction products were analyzed by Western
blotting (see FIG. 11) using the protocol described in Example 4.
The fluorescein conjugates were observed as a single band using an
anti-fluorescein primary antibody (lanes 1-4) only from the
ligation of Ph-Az with either phosphine 7 or 8, and not observed in
the case of control reactions using Ph-Q. This band was further
identified as the pIII minor coat protein by using an anti-pIII
antibody in the analysis (lanes 5-8). These results clearly show a
high degree of selectivity between phosphine 7 or 8 and the azide
containing phage peptide.
Example 7
Preservation of Phage Viability Following Selective Staudinger
Modification of a Phage-displayed Polypeptide
[0302] The present Example illustrates the preservation of phage
viability following selective Staudinger modification of a
phage-displayed polypeptide comprising p-azido-L-phenylalanine.
[0303] To show that the Staudinger coupling reaction does not lead
to a loss of infective phage particles, phage viability was
determined by titering phage Ph-Az before and after the Staudinger
ligation with 7 or 8. The observed number of viable phage particles
from a Staudinger reaction mixture was (1.7.+-.1).times.10.sup.11
plaque-forming units per milliliter (PFU/mL), compared to
(2.5.+-.1).times.10.sup.11 PFU/mL determined from control solutions
without phosphine 7 or 8.
Example 8
Phage Selection Following Staudinger Modification of a
Phage-displayed Polypeptide
[0304] The present Example illustrates the selection of phage
following the Staudinger modification of a phage-displayed
polypeptide. The selection utilized an immobilized anti-fluorescein
antibody to retain phage that comprised a conjugated phosphine 7,
which would be present only if the Staudinger modification was
successful. The phage titering also revealed phage viability.
[0305] In a model phage selection/enrichment experiment, a similar
number of phage particles prepared from the aforementioned
Staudinger ligation of phosphine 7 with Ph-Az and Ph-Q were
incubated in separate wells which were pre-coated with
anti-fluorescein antibody. After iterative washing, the bound phage
was eluted with 0.05% BSA-FITC conjugate and titered.
[0306] More specifically, anti-fluorescein antibody (20 .mu.g/mL,
250 .mu.L/well) was coated in aqueous Na.sub.2CO.sub.3 (0.1 M, pH
9.6) onto eight wells of an immuno-plate (Fisher) at 37.degree. C.
for 4 hrs. Wells were washed (3.times.0.9% NaCl-0.05% Tween 20),
blocked overnight with BSA (0.5%) at 4.degree. C., and then
incubated at room temperature for 5 hrs with phage (100 .mu.L)
either from a ligation reaction or a control solution. After being
washed, the phage was eluted from the plate with BSA-FITC conjugate
(0.05%). The recovered phage was titered.
[0307] The input phage titers were as follows: Ph-Az:
1.0.times.10.sup.9 PFU; and Ph-Q: 2.0.times.10.sup.9 PFU. The
output phage titers were Ph-Az: 1.2.times.10.sup.6 PFU; and Ph-Q:
1.0.times.10.sup.4 PFU. The recovery rate of fluorescein labeled
phage derived from the ligation of Ph-Az with 7 (0.12%) is 120 fold
greater than that of the control phage derived from Ph-Q
(0.001%).
[0308] These results from Examples 7 and 8 demonstrate that the
Staudinger ligation reaction does not significantly affect phage
viability. In contrast, it is important to note that phage Ph-Az
are nonviable after exposure to the reaction conditions in the
[3+2] cycloaddition with a terminal alkyne group and copper
catalyst. Also see, e.g., Rostovtsev et al., Angew. Chem. 2002,
114, 2708-2711; and Angew. Chem. Int. Ed. 2002, 41, 2596-2599. It
was found that the copper catalyst is predominantly responsible for
the viability loss; however, addition of high concentrations of
EDTA during the dialysis step did not notably improve phage
viability.
Example 9
Characterization of Staudinger Modification Reaction Products
[0309] To further characterize the Staudinger ligation products and
determine the conjugation efficiency, a representative Z-domain
protein (Wang et al., Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
56-61) containing p-azido-L-phenylalanine 2 at residue 7 was
expressed in an E. coli strain using mutRNA.sub.CUA.sup.Tyr (SEQ ID
NO: 1) and the mutant MjTyrRS (see, FIG. 1, SEQ ID NO: 4) that
selectively charges the tRNA with p-azido-L-phenylalanine 2 (see
Wang et al., Science 2001, 292, 498-500; Wang and Schultz, Angew.
Chem. 2005, 117, 34-68; Angew. Chem. Int. Ed. 2005, 44, 34-66; and
Chin et al., J. Am. Chem. Soc. 2002, 124, 9026-9027). Further
general description of the expression of the Z-domain polypeptide
can be found in Wang et al., Proc. Natl. Acad. Sci. U.S.A. 2003,
100, 56-61. This azide-containing Z-domain was purified and
conjugated with phosphines 7 or 8.
[0310] For this conjugation reaction, a stock (10 mM) of each
phosphine reactant was prepared in DMF, and was diluted with
reaction buffer containing mutant Z-domain protein (0.1 mM) to a
final concentration of 1 mM and total volume of 10 .mu.L. The
ligation reactions were carried out in phosphate buffered saline
solution (PBS, pH 7.4) at ambient temperature with shaking for 16
hrs. The reaction mixture was then passed through a PD-10 column,
eluted in water, dialyzed and analyzed by MALDI-TOF
spectroscopy.
[0311] The major peaks of the observed spectra match the expected
Staudinger ligation products when using the phosphine 7 as the
conjugated moiety (see FIG. 12). Peak A is assigned to the
Staudinger ligation product:
C.sub.386H.sub.556N.sub.107O.sub.119S.sub.1P.sub.1, calcd: 8662;
found: 8664. Peak B is assigned to the reduction product via
classical Staudinger reaction:
C.sub.344H.sub.529N.sub.105O.sub.110S.sub.1, calcd: 7928; found:
7928. Minor peaks a.sub.1 and b.sub.1, corresponding to A and B,
are assigned to the products derived from mutant Z-domain protein
without the first methionine. Minor peaks a.sub.2 and b.sub.2,
corresponding to A and B, are from the matrix adduct. No
azide-containing Z-domain was observable (<1%), indicating that
the reaction proceeds in high yield. For the Staudinger ligation of
phosphine 7, the conjugation efficiency is estimated to be >90%
based on the integration ratio of the peaks in the MALDI-TOF
spectrum.
[0312] FIG. 13 shows a MALDI-TOF analysis of the reaction products
from the Staudinger ligation of mutant Z-domain protein with
phosphine 8. Peak C is assigned to traceless Staudinger ligation
product: C.sub.365H.sub.539N.sub.105O.sub.116S.sub.1, calcd: 8286;
found 8286. Peak D is assigned to the reduction product via
classical Staudinger reaction:
C.sub.344H.sub.529N.sub.105O.sub.110S.sub.1, calcd: 7928; found:
7928. Peak E is assigned to the aza-ylide intermediate:
C.sub.383H.sub.552N.sub.105O.sub.117S.sub.1P.sub.1, calcd: 8562;
found: 8563. Minor peaks c.sub.1, d.sub.1 and e.sub.1,
corresponding to C, D and E, are assigned to the products derived
from mutant Z-domain protein without the first methionine. Minor
peaks c.sub.2 and d.sub.2, corresponding to C and D, are from the
matrix adduct.
[0313] In contrast to the Staudinger ligation of phosphine 7, the
traceless Staudinger ligation of phosphine 8 afforded a lower yield
of .about.50%. The lower conjugation efficiency of 8 may be due to
a slower ligation rate, presumably in the intramolecular
cyclization step (Saxon and Bertozzi, Org. Lett. 2000, 2,
2141-2143) and the ease of the hydrolysis of phenol ester in 8.
These would lead to an amine product as in the classical Staudinger
reactions (Saxon and Bertozzi, Science 2000, 287, 2007-2010; and
Staudinger and Meyer, Helv. Chim. Acta 1919, 2, 635-646).
[0314] Doping experiments with authentic material demonstrated that
the p-azido-L-phenylalanine 2 Z-domain mutant is stable. FIG. 14
provides a MALDI-TOF analysis of the reaction products from the
Staudinger ligation of p-azido-L-phenylalanine 2 containing
Z-domain protein with phosphine 7 and doping with comparative
amount of authentic p-azido-L-phenylalanine 2 Z-domain mutant.
[0315] In summary, it is shown herein that model Staudinger
ligations between fluorescein-tethered phosphines and either a
p-azido-L-phenylalanine 2 containing phage-displayed peptide or a
mutant Z-domain protein occur with excellent selectivity and
efficiency. The Staudinger ligation does not affect phage viability
so that after the completion of ligation enrichment can be
performed without difficulty. This work provides useful methods for
selectively modifying proteins without altering their function and
should be useful for the generation of highly homogenous PEGylated
proteins, surface immobilized proteins or proteins modified with
spectroscopic or affinity reagents.
[0316] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended
claims.
[0317] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. For example, all the
techniques and apparatus described above can be used in various
combinations. All publications, patents, patent applications,
and/or other documents cited in this application are incorporated
by reference in their entirety for all purposes to the same extent
as if each individual publication, patent, patent application,
and/or other document were individually indicated to be
incorporated by reference for all purposes.
Sequence CWU 1
1
15 1 77 RNA Artificial Sequence mutant Methanococcus jannaschii
suppressor tyrosyl-tRNA-CUA 1 ccggcgguag uucagcaggg cagaacggcg
gacucuaaau ccgcauggcg cugguucaaa 60 uccggcccgc cggacca 77 2 306 PRT
Methanococcus jannaschii 2 Met Asp Glu Phe Glu Met Ile Lys Arg Asn
Thr Ser Glu Ile Ile Ser 1 5 10 15 Glu Glu Glu Leu Arg Glu Val Leu
Lys Lys Asp Glu Lys Ser Ala Tyr 20 25 30 Ile Gly Phe Glu Pro Ser
Gly Lys Ile His Leu Gly His Tyr Leu Gln 35 40 45 Ile Lys Lys Met
Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile 50 55 60 Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp 65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met 85
90 95 Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp
Lys 100 105 110 Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys 115 120 125 Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro 130 135 140 Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Asp Ile His 145 150 155 160 Tyr Leu Gly Val Asp Val
Ala Val Gly Gly Met Glu Gln Arg Lys Ile 165 170 175 His Met Leu Ala
Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His 180 185 190 Asn Pro
Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser 195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala 210
215 220 Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn
Pro 225 230 235 240 Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro
Leu Thr Ile Lys 245 250 255 Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr
Val Asn Ser Tyr Glu Glu 260 265 270 Leu Glu Ser Leu Phe Lys Asn Lys
Glu Leu His Pro Met Asp Leu Lys 275 280 285 Asn Ala Val Ala Glu Glu
Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys 290 295 300 Arg Leu 305 3
918 DNA Methanococcus jannaschii 3 atggacgaat ttgaaatgat aaagagaaac
acatctgaaa ttatcagcga ggaagagtta 60 agagaggttt taaaaaaaga
tgaaaaatct gcttacatag gttttgaacc aagtggtaaa 120 atacatttag
ggcattatct ccaaataaaa aagatgattg atttacaaaa tgctggattt 180
gatataatta tattgttggc tgatttacac gcctatttaa accagaaagg agagttggat
240 gagattagaa aaataggaga ttataacaaa aaagtttttg aagcaatggg
gttaaaggca 300 aaatatgttt atggaagtga attccagctt gataaggatt
atacactgaa tgtctataga 360 ttggctttaa aaactacctt aaaaagagca
agaaggagta tggaacttat agcaagagag 420 gatgaaaatc caaaggttgc
tgaagttatc tatccaataa tgcaggttaa tgatattcat 480 tatttaggcg
ttgatgttgc agttggaggg atggagcaga gaaaaataca catgttagca 540
agggagcttt taccaaaaaa ggttgtttgt attcacaacc ctgtcttaac gggtttggat
600 ggagaaggaa agatgagttc ttcaaaaggg aattttatag ctgttgatga
ctctccagaa 660 gagattaggg ctaagataaa gaaagcatac tgcccagctg
gagttgttga aggaaatcca 720 ataatggaga tagctaaata cttccttgaa
tatcctttaa ccataaaaag gccagaaaaa 780 tttggtggag atttgacagt
taatagctat gaggagttag agagtttatt taaaaataag 840 gaattgcatc
caatggattt aaaaaatgct gtagctgaag aacttataaa gattttagag 900
ccaattagaa agagatta 918 4 306 PRT Artificial Sequence
p-azido-L-phenylalanine aminoacyl-tRNA synthetase clone-1 amino
acid sequence (derived from wild-type Methanococcus jannaschii
tyrosyl tRNA-synthetase) 4 Met Asp Glu Phe Glu Met Ile Lys Arg Asn
Thr Ser Glu Ile Ile Ser 1 5 10 15 Glu Glu Glu Leu Arg Glu Val Leu
Lys Lys Asp Glu Lys Ser Ala Thr 20 25 30 Ile Gly Phe Glu Pro Ser
Gly Lys Ile His Leu Gly His Tyr Leu Gln 35 40 45 Ile Lys Lys Met
Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile 50 55 60 Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp 65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met 85
90 95 Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Asn Phe Gln Leu Asp
Lys 100 105 110 Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys 115 120 125 Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro 130 135 140 Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Pro Leu His 145 150 155 160 Tyr Gln Gly Val Asp Val
Ala Val Gly Gly Met Glu Gln Arg Lys Ile 165 170 175 His Met Leu Ala
Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His 180 185 190 Asn Pro
Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser 195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala 210
215 220 Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn
Pro 225 230 235 240 Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro
Leu Thr Ile Lys 245 250 255 Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr
Val Asn Ser Tyr Glu Glu 260 265 270 Leu Glu Ser Leu Phe Lys Asn Lys
Glu Leu His Pro Met Asp Leu Lys 275 280 285 Asn Ala Val Ala Glu Glu
Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys 290 295 300 Arg Leu 305 5
306 PRT Artificial Sequence p-azido-L-phenylalanine aminoacyl-tRNA
synthetase clone-2 amino acid sequence (derived from wild-type
Methanococcus jannaschii tyrosyl tRNA-synthetase) 5 Met Asp Glu Phe
Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser 1 5 10 15 Glu Glu
Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr 20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln 35
40 45 Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile
Ile 50 55 60 Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly
Glu Leu Asp 65 70 75 80 Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys
Val Phe Glu Ala Met 85 90 95 Gly Leu Lys Ala Lys Tyr Val Tyr Gly
Ser Ser Phe Gln Leu Asp Lys 100 105 110 Asp Tyr Thr Leu Asn Val Tyr
Arg Leu Ala Leu Lys Thr Thr Leu Lys 115 120 125 Arg Ala Arg Arg Ser
Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro 130 135 140 Lys Val Ala
Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Ser His 145 150 155 160
Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile 165
170 175 His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile
His 180 185 190 Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met
Ser Ser Ser 195 200 205 Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro
Glu Glu Ile Arg Ala 210 215 220 Lys Ile Lys Lys Ala Tyr Cys Pro Ala
Gly Val Val Glu Gly Asn Pro 225 230 235 240 Ile Met Glu Ile Ala Lys
Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys 245 250 255 Arg Pro Glu Lys
Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu 260 265 270 Leu Glu
Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys 275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys 290
295 300 Arg Leu 305 6 306 PRT Artificial Sequence
p-azido-L-phenylalanine aminoacyl-tRNA synthetase clone-3 amino
acid sequence (derived from wild-type Methanococcus jannaschii
tyrosyl tRNA-synthetase) 6 Met Asp Glu Phe Glu Met Ile Lys Arg Asn
Thr Ser Glu Ile Ile Ser 1 5 10 15 Glu Glu Glu Leu Arg Glu Val Leu
Lys Lys Asp Glu Lys Ser Ala Thr 20 25 30 Ile Gly Phe Glu Pro Ser
Gly Lys Ile His Leu Gly His Tyr Leu Gln 35 40 45 Ile Lys Lys Met
Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile 50 55 60 Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp 65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met 85
90 95 Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp
Lys 100 105 110 Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys 115 120 125 Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro 130 135 140 Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Pro Leu His 145 150 155 160 Tyr Gln Gly Val Asp Val
Ala Val Gly Gly Met Glu Gln Arg Lys Ile 165 170 175 His Met Leu Ala
Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His 180 185 190 Asn Pro
Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser 195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala 210
215 220 Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn
Pro 225 230 235 240 Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro
Leu Thr Ile Lys 245 250 255 Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr
Val Asn Ser Tyr Glu Glu 260 265 270 Leu Glu Ser Leu Phe Lys Asn Lys
Glu Leu His Pro Met Asp Leu Lys 275 280 285 Asn Ala Val Ala Glu Glu
Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys 290 295 300 Arg Leu 305 7
306 PRT Artificial Sequence p-azido-L-phenylalanine aminoacyl-tRNA
synthetase clone-4 amino acid sequence (derived from wild-type
Methanococcus jannaschii tyrosyl tRNA-synthetase) 7 Met Asp Glu Phe
Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser 1 5 10 15 Glu Glu
Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu 20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln 35
40 45 Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile
Ile 50 55 60 Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly
Glu Leu Asp 65 70 75 80 Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys
Val Phe Glu Ala Met 85 90 95 Gly Leu Lys Ala Lys Tyr Val Tyr Gly
Ser Thr Phe Gln Leu Asp Lys 100 105 110 Asp Tyr Thr Leu Asn Val Tyr
Arg Leu Ala Leu Lys Thr Thr Leu Lys 115 120 125 Arg Ala Arg Arg Ser
Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro 130 135 140 Lys Val Ala
Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Val His 145 150 155 160
Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile 165
170 175 His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile
His 180 185 190 Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met
Ser Ser Ser 195 200 205 Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro
Glu Glu Ile Arg Ala 210 215 220 Lys Ile Lys Lys Ala Tyr Cys Pro Ala
Gly Val Val Glu Gly Asn Pro 225 230 235 240 Ile Met Glu Ile Ala Lys
Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys 245 250 255 Arg Pro Glu Lys
Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu 260 265 270 Leu Glu
Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys 275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys 290
295 300 Arg Leu 305 8 306 PRT Artificial Sequence
p-azido-L-phenylalanine aminoacyl-tRNA synthetase clone-5 amino
acid sequence (derived from wild-type Methanococcus jannaschii
tyrosyl tRNA-synthetase) 8 Met Asp Glu Phe Glu Met Ile Lys Arg Asn
Thr Ser Glu Ile Ile Ser 1 5 10 15 Glu Glu Glu Leu Arg Glu Val Leu
Lys Lys Asp Glu Lys Ser Ala Ala 20 25 30 Ile Gly Phe Glu Pro Ser
Gly Lys Ile His Leu Gly His Tyr Leu Gln 35 40 45 Ile Lys Lys Met
Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile 50 55 60 Leu Leu
Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp 65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met 85
90 95 Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Arg Phe Gln Leu Asp
Lys 100 105 110 Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr
Thr Leu Lys 115 120 125 Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg
Glu Asp Glu Asn Pro 130 135 140 Lys Val Ala Glu Val Ile Tyr Pro Ile
Met Gln Val Asn Val Ile His 145 150 155 160 Tyr Asp Gly Val Asp Val
Ala Val Gly Gly Met Glu Gln Arg Lys Ile 165 170 175 His Met Leu Ala
Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His 180 185 190 Asn Pro
Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser 195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala 210
215 220 Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn
Pro 225 230 235 240 Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro
Leu Thr Ile Lys 245 250 255 Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr
Val Asn Ser Tyr Glu Glu 260 265 270 Leu Glu Ser Leu Phe Lys Asn Lys
Glu Leu His Pro Met Asp Leu Lys 275 280 285 Asn Ala Val Ala Glu Glu
Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys 290 295 300 Arg Leu 305 9
306 PRT Artificial Sequence p-azido-L-phenylalanine aminoacyl-tRNA
synthetase clone-6 amino acid sequence (derived from wild-type
Methanococcus jannaschii tyrosyl tRNA-synthetase) 9 Met Asp Glu Phe
Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser 1 5 10 15 Glu Glu
Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Gly 20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln 35
40 45 Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile
Ile 50 55 60 Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly
Glu Leu Asp 65 70 75 80 Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys
Val Phe Glu Ala Met 85 90 95 Gly Leu Lys Ala Lys Tyr Val Tyr Gly
Ser Thr Phe Gln Leu Asp Lys 100 105 110 Asp Tyr Thr Leu Asn Val Tyr
Arg Leu Ala Leu Lys Thr Thr Leu Lys 115 120 125 Arg Ala Arg Arg Ser
Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro 130 135 140 Lys Val Ala
Glu Val Ile Tyr Pro Ile Met Gln Val Asn Thr Tyr Tyr 145 150 155 160
Tyr Leu Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile 165
170 175 His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile
His 180 185 190 Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met
Ser Ser Ser 195 200 205 Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro
Glu Glu Ile Arg Ala 210 215 220 Lys Ile Lys Lys Ala Tyr Cys Pro Ala
Gly Val Val Glu Gly Asn Pro 225 230 235 240 Ile Met Glu Ile Ala Lys
Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys 245 250 255 Arg Pro Glu Lys
Phe Gly Gly
Asp Leu Thr Val Asn Ser Tyr Glu Glu 260 265 270 Leu Glu Ser Leu Phe
Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys 275 280 285 Asn Ala Val
Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys 290 295 300 Arg
Leu 305 10 306 PRT Artificial Sequence p-azido-L-phenylalanine
aminoacyl-tRNA synthetase clone-7 amino acid sequence (derived from
wild-type Methanococcus jannaschii tyrosyl tRNA-synthetase) 10 Met
Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser 1 5 10
15 Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu
20 25 30 Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr
Leu Gln 35 40 45 Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe
Asp Ile Ile Ile 50 55 60 Leu Leu Ala Asp Leu His Ala Tyr Leu Asn
Gln Lys Gly Glu Leu Asp 65 70 75 80 Glu Ile Arg Lys Ile Gly Asp Tyr
Asn Lys Lys Val Phe Glu Ala Met 85 90 95 Gly Leu Lys Ala Lys Tyr
Val Tyr Gly Ser Pro Phe Gln Leu Asp Lys 100 105 110 Asp Tyr Thr Leu
Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys 115 120 125 Arg Ala
Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro 130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gln Ile His 145
150 155 160 Ser Ser Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg
Lys Ile 165 170 175 His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val
Val Cys Ile His 180 185 190 Asn Pro Val Leu Thr Gly Leu Asp Gly Glu
Gly Lys Met Ser Ser Ser 195 200 205 Lys Gly Asn Phe Ile Ala Val Asp
Asp Ser Pro Glu Glu Ile Arg Ala 210 215 220 Lys Ile Lys Lys Ala Tyr
Cys Pro Ala Gly Val Val Glu Gly Asn Pro 225 230 235 240 Ile Met Glu
Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys 245 250 255 Arg
Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu 260 265
270 Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285 Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile
Arg Lys 290 295 300 Arg Leu 305 11 10 PRT Artificial Sequence pIII
fusion streptavidin binding peptide (SBP) MISC_FEATURE (3)..(3) X
is an unnatural amino acid 11 Ala Gly Xaa Thr Leu Leu Ala His Pro
Gln 1 5 10 12 39 DNA Artificial Sequence FT18 PCR primer 12
gacagcttat catcgatgag acgttgatcg gcacgtaag 39 13 40 DNA Artificial
Sequence FT19 PCR primer 13 ggttggtttg cgcattcagc gctaaccgtt
tttatcaggc 40 14 25 DNA Artificial Sequence FT121 primer 14
catgcccggg tacctttcta ttctc 25 15 79 DNA Artificial Sequence FT126
template 15 catgtttcgg ccgagccccc accctgcgga tgagccagca aagtctagcc
ggcagagtga 60 gaatagaaag gtacccggg 79
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